Comprehensive Overview of the Immunohistochemistry (IHC) Process

[Music] now I'm going to discuss in details about the ISC process which is basically

carried out in our Institute and what are the basic steps so remember one very important thing okay everyone please

remember this thing that you or whatever process that is carried out in your Institute it might vary from what is

carried in our Institute and it varies from one place to another place from country to Country even between the

different states it might vary even in the same state different colleges might have different protocol okay so this is

the one that we follow in our Institute you have to write the process that you follow in your Institute more or less

all the ISC process and the steps they are the same across all the institutes only there is some change in the type of

buffer you are using the pH that you're using and the time and the duration is different in the different institutes

else everything or the basic concept is the same so you have taken the the formal and fixed paraffin embedded

tissue cut the sections out of it you have taken it basically you have to De parapan IE so how do you de parapan

eyesee you put in xylene two changes each for 5 minutes then you have to have to rehydrate how do you rehydrate you

are going to first keep in 100% alcohol then 70% and 50% each for 5 minutes each for 5 minutes you are keeping then you

wash and tap water to remove extra amount of alcohol or excess alcohol after after that you wash with distal

water for 2 minutes once okay okay then we go for antigen retrial technique so the antigen retrieval solution that we

use is the Tris EDTA buffer at a pH of 9.0 okay in the microwave you have to carry out three changes each of 5

minutes if you're using a pressure cooker normal pressure cooker then two whistles is enough then you cool the

slides gradually and then put the slide in wash buffer so instead of a normal TBS as you have seen in the in many

textbook they are using TBS but we are using a wash buffer basically the pH of which is is adjusted between 7 to 7.4

okay this is the wash buffer so after that we are going to go for the peroxidase block we have to block the

endogenous peroxidase activity so add peroxidase block to the slide and incubate for 10 minutes at the room

temperature so what is the peroxides block we have discussed in details uh already so I'm not going to details

about that after that you have to add the protein block so you can use the normal serum or you can use the Bine

albumin as well to prevent any background staining and incubate for 10 minutes at the room temperature then we

add the primary antibody so add the primary antibody and incubate for 30 to 45 minutes at room temperature then the

slides again you put the slides in the wash buffer two changes each for 3 minutes okay then you add the secondary

antibodies so over here you are going to add the complex the poly hos radish peroxidase complex is added and

incubated for 20 minutes at room temperature so uh these timings that we are seeing it will vary from Institute

to Institute and after that after you put the secondary antibody again you wash for two changes each for 3 minutes

okay then after that you are have to add the chromogen that is diaminobenzidine that is dab reagent so the dab basically

how it is prepared you have to add 1 ml dab buffer to one drop of dab so 1 ml of this is added to one drop of this

reagent and then basically after you add the chromogen you again put the particular slide in the wash buffer one

change for 2 minutes and then you put the slides in distilled water okay after this you have to do the counter staining

so you do counter staining with hematoxilin for 20 to 30 seconds then you wash the slide under the running tap

water okay now over here the your work is complete everything is done then you will carry out dehydration alcohol chain

so first we had rehydrated so over here we are going to dehydrate so dehydrated so from 50 uh uh 50% 70% 100% each for 2

minutes you have to keep okay to dehydrate then we have to do the clearing so clearing is done in xylene

two changes each for 1 minute and after that you mount the slide with the DPX okay so with this we have basically

completed the entire ITC process and the procedure which is carried out in our Institute and this is the of part one

okay part one of our iy in the next lecture we will discuss the troubleshooting as well as we are also

going to discuss about the different individual iy markers which are basically used so thank you very much

for watching this particular video myself Dr jiad presents to you simply pathology and today we are back with a

very important session today as you can see we are going to read about the imunohistochemistry or imuno

cytochemistry so what is the difference between imuno hiso and immunocytochemistry so this IC is

basically carried out in formalin fixed paraffin embedded tissue blocks okay so it is basically carried out in the

tissues okay in the tissues okay in the tissue specimen whereas the immunocyto chemistry is basically carried out in on

the cellular sample okay on the cells directly for example the fnc material or it is carried out on Cell Block okay

cell block okay okay so but the basic principle of both ITC as well as imuno phoch chemistry they are the same that

is why we are discussing both of them together so what is it that we are going to discuss in today's topic of

discussion so first and foremost we are going to understand the basic basic Immunology okay basic Immunology okay

because if you don't know the basic Immunology you will not be able to appreciate the principle okay next we

are going to see the IC principle okay IC principle then we are going to see the various methods okay the various

methods or the various principles of carrying out the imunohistochemistry or immunocytochemistry after that we are

going to see the the procedure of carrying out this process both immunocytochemistry as well as

imunohistochemistry procedure okay in detail so we are going to see and we are also going to see the procedure which we

are seeing in our Institute so this is basically part one of this IC series whereas in part two we are going to

cover the troubleshooting part okay what is troubleshooting or some of the problems that you are facing while

carrying out the imunohistochemistry and along with that most importantly we are going to see the different IC markers

okay different IC markers okay so this will be covered in part two okay this will be covered in part

two okay so let us begin today's topic of discussion without wasting any time so ISC or immunocytochemistry okay it is

a technique to visualize recognition of antigen which is present in the tissue with the help of corresponding antibody

okay so you are detecting an antigen with the help of an antibody so IC technique was successfully introduced in

routine formalin fixed paraffin embedded section tissue sections by tayor and burns in

1974 subsequently the development of monoclonal antibody introduced a new era in the imunohistochemistry however it

took another 10 to 15 years to have regular routine use of IC in pathology diagnostic laboratory presently

imunohistochemistry or IC it is an essential technique in every pathology laboratory now before we go into the

details we will have to understand the basic immunology and why basic Immunology is necessary why so before we

go into the details we have to understand some things so what is the basic principle what is the basic

principle of immunocytochemistry or imunohistochemistry so what what is the basic principle the basic principle of

IC or ICC it is to demonstrate the presence of a specific antigen in the Cell by applying corresponding antibody

to have antigen antibody reaction so basically what is here over here this is a particular tissue and

this tissue is having a certain antigen as you can appreciate now this particular antigen is basically binding

with a particular antibody so this antibody is going to bind with this particular antigen only if the

corresponding antigen is present okay so there will be antigen antibody reaction if that particular antigen is not

present the antibody is not going to bind and therefore the reaction will be negative so this particular antigen it

is binding with the antibody with the help of a particular site okay that is called as antigenic determinant site I

am going to repeat this once again so antibody is going to bind with the antigen okay but the antibody is binding

to the antigen at a particular site this site this site where the antibody is binding to the antigen it is called as

an epitope or it is called as antigenic determinant site so it is called as an epitope or antigenic determinant site

remember one thing a single antigen can have multiple epitopes therefore multiple sides of binding of different

kinds of antibody can be there in a single antigen okay so just remember this concept okay so the antigen

contains an epitope or antigenic determinant site okay that is this is the epitope this part is the epitope or

the antigen determinant site that evokes a specific immunological response to develop

antibody this epitope or this antigenic determinant site this is basically inducing the formation of a particular

antibody which will bind to this particular epitope or this antigenic determinant site the antigen epitope

site and the antibody binding site they have complimentary geometrical shape so as you can see what is the meaning of

complimentary geometric shape compliment geometric shape means that see this particular antigen determined side this

is v-shaped whereas this antibody if you see this is little bit outward so it is fitting into the V of the antigen so

they are having complimentary geometrical and chemical feature this is responsible for the antigen antibody

reaction this antigen antibody reaction is further visualized by attaching a certain label to the primary or the

secondary antibody and which basically hydes a particular substrate giving a color which is then seen under the

microscope okay so this is the basic principle of imunohistochemistry or immunocytochemistry so after having

known this we uh you know we have to understand certain important key terms in the basic Immunology so what is an

antigen so these are very important vaa questions that are asked in the exam so what is an antigen and any substance

that is capable of producing an immunological or immunogenic response is called called an antigen now there are

specific set of chemical components that evoke immunogenic response of the antigen which is known as epitope or

antigenic determinant site which we have already spoken and seen okay what is antigenic determinant site or the

epitope similarly we are having just like there is an antigen so there is an antibody so we have read and seen in

details about this antibody where we have already read about this in the topic of immune system in the topic of

immunity okay this is just a revision antibody is also known as an imunoglobulin the antibody is produced

by plasma cells in response to antigenic stimulation imunoglobulin has a specific Affinity against the epitope of the

antigen as I told you an imunoglobulin or an antibody they will have affinity for the antigenic determinant site or

the epitope which has induced its formation each imunoglobulin if you see they are composed of a pair of light

chain so this is the light chain as you can appreciate okay there is a pair of light chain and and there is a pair of

heavy chain we can see light chain as well as we can see heavy chain this light chain as we can appreciate over

here they are of two type they are Kaa or Lambda whereas there are five types of the heavy chain what are the

different five types they can be Alpha Gamma Delta Epsilon mu and depending on the nature of the heavy chain which is

present the imunoglobulin will be called as IG g a g d e or M respectively now the antibody is a y shaped structure as

we can appreciate the two tips of the Y are known as antigen binding site so as you can appreciate this is the antibody

if you look at the tip of this particular antibody in the light chain over here is what is known as the

antigen binding site of the imunoglobulin now the base of each arm of the Y is the hinge region as we can

appreciate this is the Hing region it is the flexible region of the imunoglobulin so this is how an antibody looks like

okay next now what is a hybridoma technique so it is a technique okay where abundant unlimited amount of pure

homogenous immunog globulin is produced now the antibody producing B lymphocyte it is fused with a malignant

Immortal plasma cell okay so a malignant Immortal plasma cell is fusing with this to form what is called as a hybrid cell

and this hybrid cell this resultant hybrid cell it acquires the capability of unlimited proliferation and

production of a specific antibody the resulting antibody in this condition is known as monoclonal antibody so remember

when only a single group of cell okay or related group of cells are producing antibodies it is called as monoclonal

antibody whereas what is the concept of polyclonal when multiple different B lymphocytes are producing antibodies so

their whole antibody together they form what is known as polyclonal antibody okay so over here since a single B

lymphocytic bodies and they are having unlimited potential of producing unlimited number of antibodies such

antibodies they are monoclonal in nature so they are named as monoclonal because they are produced from a single clone of

cell each hybridoma cell produces only a specific antibody which is specific for a particular antigen okay so this is the

monoclonal anti antibody then we have the concept of polyclonal antibody which is generated from a different B

lymphocytopenia generated from the different B lymphocytes in response to different epitopes of a single antigen

so just remember one thing they are generated from different B lymy not a single B lymy and for example this is

particular antigen okay now this antigen this can be one epitope this can be epitope number two epitope number three

epitope number four okay so over here what happens in polyclonal antibod so this particular epitope okay E1 this is

inducing B1 lymphocytic body against this similarly this is E2 epitope which is stimulating a different B

lymphocytes a different kind of antibody against E2 similarly E3 epitope E4 epitope E5 epitope in the different

antigens they are inducing different sets of V lympho side to int you know to make antibody against a different

epitopes so so basically what happens that polyclonal antibody is generated from different B lymphocytes in response

to different epitopes of a single antigen as they are generated from the different clone of B cells these

antibodies are called polyclonal in nature there is a chance of batch to batch variation in case of polyclonal

antibody so basically at this point just try to understand so whenever you're using a monoclonal antibody okay while

carrying out imunohistochemistry okay you will get consistent results okay whereas if you use polyclonal antibod so

from one batch to another bad the results might vary okay so that is why this is very important monoclonal

antibodies they are better and more expensive as compared to polyclonal antibodies okay so I will come into more

details about what monoclonal and what polyclonal antibody is and what is the difference between the two okay so

before that we will learn certain terms what is avidity avidity means the overall functional strength of binding

capacity of antibody and antigen complex so with what strength they are antigen antibody they are binding with each

other that is the avidity the polyclonal antibody reacts with multiple epitope sides of an an anen right and therefore

the overall strength of antigen antibody complex is very strong when a polyclonal antibody is used okay so as I told you

over here if you see the monoclonal antibody if you see over here only a single B

lymphocytic bodies against a certain antigen okay for example this is antigen and this is one epitope of the antigen

so over here monoclonal uh B cells or single group or single clone of B cells are only producing an antibody against

only a single single epitope okay so these are highly specific monoclonal antibodies are highly specific okay but

for example they are not very sensitive because they are directed against only a single epitope so as a result the

monoclonal antibodies will bind with only one epitope and therefore the antigen antibody reaction is not very

strong whereas as I told you that in case of polyclonal uh B cell if you see that there are multiple polyclonal B

cells which are directed against multiple epitopes of a particular antigen so over there is E1 E2 E3 so the

polyclonal group of of antibodies okay they are not very specific for a particular epitope but what happens over

here that they because a multiple number of antibodies can bind multiple epitopes of the antigen so the antigen antibody

bond is very strong in case of polyclonal antibody you know in case we are using polyclonal antibody so the

avidity of an antibody depends on following factors the more the valency of the antibody the greater is the

avidity the Affinity the Affinity between the individual epitope of the antigen and the corresponding antigen

binding site of the antibody okay and the structural rearrangement also depend the avidity will also depend on the

three-dimensional structural arrangement of the antigen as well as the antibody okay okay so now we are going to see the

difference between the monoclonal and polyclonal antibodies so monoclonal antibod they are very high production

cost because over here you have to bring in the two cells together and then you have to fuse them and then you have to

induce immortality in the B cell to produce unlimited number of antibodies against a single epitope of a particular

antigen so therefore specialized training is required in monoral antibody it is directed against a particular

epitope of an antigen so only one type of B cell producing antibody against a particular epitope if this is an antigen

only against this one epitope the antibody is produced and only same types of B cells acting against a single

epitope so these are monoclonal in nature there is no variation of batch to batch so whatever results you get for

ISC there is no variation as such because these are all monoclonal antibodies so there is a stability of

the in the in the results okay from batch to batch they have a very high specificity because whatever antibody

they are producing they are highly specific for only a single epitope okay only against a single epitope but they

are less robust for detection as antibody is directed to a single epitope so what happens since they are highly

specific then they are only going to bind with a particular epitope of an antigen so what happens that uh uh they

cannot detect the antibody antigen reaction very smoothly okay so basically they you know the antigen antibody

reaction is not very robust in case of monoclonal antibody whereas in polyclonal antibody there is there's a

lowc cost production okay it is not a highly specialized process and no special training is required to produce

so over here multiple clones of B cells are there okay which are directed again which are directed against multiple

epitopes of a particular antigens this is epitope one this is epitope 2 this is epitope 3 so what happens that all of

them are producing antibodies which can which will go and combined with the respective epitopes so over here what

happens that they are directed to multiple epitopes of a particular antigen therefore the results okay the

results are not very specific so they are having a low specificity and therefore the results also varies from

batch to batch but very importantly they have a very uh you know strong antigen antibody

interaction why because multiple antibodies directed against multiple epitopes are produced so the antigen

antibody binding is very strong so more robust in detection as the antibodies are directed against multiple epitopes

so I hope you have understood the concept of monoclonal antibody and polyclonal

antibody now we are going to understand the term what is antibody specificity it indicates the the precise detection of a

specific epitope of the antigen by the antibody a particular antigenic determinant side that is the epitope can

be present in more than one antigen as I've already shown you and therefore a single antibody May react with different

antigens okay always remember this point okay so a particular epitope can be present in more than one antigen

therefore a single antibody May react with different antigens as well so if the monoclonal antibody as I told you

they are they are acting against only a single epitope okay so that is why monoclonal antibodies are highly

specific whereas the polyclonal antibodies if you see they are acting against different epitopes of the same

antigen therefore they are not specific and what is the sensitivity therefore remember the monoclonal antibodies are

highly specific whereas the polyal antibodies they are not at all specific they have less specific okay whereas

what is the sensitivity it is related to the detection of the relative amount of antigen by the antibody in a particular

technique the highly sensitive technique detects even the low amount of antigen and the relative intensity of the signal

of antigen antibody reaction is much strong so now you people tell me sensitivity will be more with either

monoclonal or with polyclonal yes what will be the answer so as I told you as I told you that basically if this is

particular antigen okay and if we are using a monoclonal antibody which is directed against only a single

particular epitope now if this the amount of antigen is very less then what is going to happen because the

monoclonal antibodies are highly specific therefore the reaction between antigen antibod will be very less

because they will bind with only a very specific epitope so that is why the sensitivity with monoclonal antibodies

becomes less the chances of detection becomes less with monoclonal whereas with polyclonal antibody as I told you

if you use polyclonal antibodies so there are multiple antibodies directed against multiple epitopes of the same

antigen right so as a result what happens that the antigen antibody reaction is very strong so with polycal

antibodies you can easily detect even very low amount of antigens because they have a strong antigen antibody reaction

so I hope this basic concept is very crystal clear to everyone because this is most important for the understanding

of the basic principle of the imunohistochemistry okay so what is sensitivity what is specificity

antibody specificity what is avidity what is monoclonal antibody what is polyclonal antibody what is an antibody

and what is an antigen so these are very very important basic okay points that are asked in the exam so having

completed the basic principle of the immunochemistry now we are going to see the different kinds of detection system

so it is not possible to detect the antigen antibody reaction by light microscope now so for example if you

have have reacted okay one antigen and one antibody with each other and if you see under the microscope you cannot

understand what is happening so you need a suitable detection system or visualization system in necessary for

demonstration of such a reaction so the different types of the detection systems are highlighted now which are very

important and they are asked in the exam so usually there are two kinds of methods one is direct one is indirect

and again indirect methods they are you know superseded by some other method so what is the direct method very simple so

over here in the direct method the primary antibody this is the primary antibody okay this is a primary antibody

it is directed uh it is directly tagged with an enzyme of floresent so you can see this something blue this is actually

a label they are labeled by either an enzyme or by flosin so when the primary antibody will bind with the antigen then

in that case we will add a a particular chromogen and that chromogen in presence of this particular enzyme of fluoresence

is basically going to flues or it will give out a color okay and that will be detected and seen under the microscope

okay so this is is completely direct so over here the antibody that we use they should be specific for the particular

antigen otherwise non-specific straining may occur so for example this is the direct method over here this is the

particular tissue and this is the antigen in question so over here the primary antibody will come and bind to a

particular antigen okay against which it is directed okay and this primary antibody is labeled with a particular

enzyme so what happens that once the antigen antibody is going to bind with each other then only this particular

enzyme okay is going to you know because of the binding the enzyme will be there and once you add the chromogen that is

going to give the color and then it will be detected but one very important thing over here is that the primary antibody

should be highly specific for the antigen if the primary antibody is binding with the antigen which is under

question and also with certain other normal antigens if there is a non-specific binding then it is going to

give false positive reaction so the advantage of this direct method it is a rapid and a simple method but what is

the disadvantage so over here in the direct method different primary antibodies should be labeled differently

for the antigen and it has a low sensitivity so different primary antibodies should be labeled differently

for the antigen so for example if you are you know uh you have to use multiple primary antibodies over here in this

direct method okay so this is one disadvantage of this method now coming to the indirect method so over here what

is happening in the indirect method in the indirect conjugated method the primary antibody is unlabeled we do not

uh you know label the primary antibod so this is the primary antibody which is shown in basically uh red it is not

labeled we do not have to label the primary antibody unlike in the direct method we use instead a secondary

antibody is conjugated and is directed against the primary antibod so secondary antibody is basically directed against

the primary antibody and this secondary antibody if you see okay it is basically containing a particular enzyme or you

know so basically this basically has this enzyme system so this antigen this is the antigen this antigen primary

antibody secondary antibody complex can be visualized by a suitable chromogen okay so this is the indirect method now

what is the advantage over here a single conjugated secondary antibody can be used against different primary

antibodies so you can use different types of secondary antibody against different primary antibodies so you

don't have to use multiple primary antibod this is one advantage again the primary uh the primary anti antibody

because over here uh we are having a secondary antibody also so basically higher dilution of primary antibody can

be used over here okay large amount of secondary antibody can be easily produced against the primary antibody so

it is much easier to produce secondary antibody against primary then producing primary antibody against different

antigens so over here we can use less amount of primary antibody okay so higher dilution can be used and large

amounts of secondary antibody can be easily produced against the primary antibody and for negative control the

primary antib body can just be omitted this is how we use a negative control this is the indirect method okay these

are the two very simple methods after that we are having certain very important processes that is the

peroxidase antiperoxidase method this is some kind of indirect method only so what happens over here just just

remember this is also an indirect method so there is a particular antigen there's a primary antibody okay and there's a

secondary antibody but this secondary antibody it is not directly attached to a particular chromogen instead it is

attached to a particular complex so what is this complex this complex is in itself is having a enzyme anti-enzyme

complex so there's a peroxidase antiperoxidase complex is there so let us try to understand what it is the

peroxidase antiperoxidase reagent it is an immune complex substance as you can appreciate this is the peroxidase

antiperoxidase complex it is an immune complex substance and it is containing a particular hor radish peroxidase antigen

so this is basically the H radish peroxides hrp that we call it okay this is the horse radish peroxidase antigen

or this is the particular peroxidase enzyme and this particular peroxidase enzyme if you see it is basically joined

or it is forming a complex with antibody against it that that that is with antiperoxidase so there is a complex

between peroxidase and anti peroxidases so over here we can see three enzymes over here which is forming a complex

with the corresponding antibodies as we can appreciate and which is called as peroxidase antiperoxidase

complex here remember a secondary bridging antibody is used between the primary and the peroxid anti peroxidase

complex so basically this secondary antibody which is acting as a bridging antibody between the primary and the

peroxidase antiperoxidase complex now the primary antibody and the peroxidase antiperoxidase reagents they are from

the same species as you can see this is also pink in color this is also pink in color so both the primary and the

peroxidase anti peroxidase reagent they are from the same species whereas the secondary linking antibody is from a

different species this secondary antibody is usually highly specific against the primary antibody and the

antibody present in the peroxidase antiperoxidase complex so this secondary antibody is highly specific not only it

is highly specific for the primary antibody but it is highly specific for the peroxid anti peroxidase complex so

you must be thinking why we are using this peroxid antioxid complex try to understand over here because we are

using a greater number of enzyme markers or labels so in this system what is happening over here you can see that

that for a single antigen we are using three such peroxides or three enzymes are there if we add chromogen then

basically all these three enzyme is going to metabolize that particular chromogen and release the color and the

color will be generated so because more amount of labels are there so more amount of color will be generated so the

importance is that even if a very small amount of antigen is present they will be highlighted because of this

peroxidase antiperoxidase complex so what happens that with this peroxidase antiperoxidase system actually both of

them are same in the indirect method also in the indirect method if you see this is also acting as a bridge between

this particular enzyme and this particular primary antibody over here also it is the same but the only thing

is instead of using one enzyme I'm using a complex of enzymes so the reaction will be more easily seen so because of

this peroxid anterior peroxid system it has a high degree of sensitivity the method is thousand times more sensitive

than this indirect conjugate method this is the indirect method so in compar with this this is 100 times more uh sensitive

why it is more because for a single particular antigen we are using three particular you know enzyme that is label

that is the peroxidase which is going to you know metabolize the particular chromogen and give out the color okay so

this is the very very important point hope you have understood this first method then comes the second method that

is the avidin biotin method what is this method now over here again the same thing so the so over here remember we

are having two things one is the Biotin and one is the aiin normally this biotin is having a very high affinity for

avidin okay so in this method the secondary antibody is tagged with biotin so this is that is in red this is the

secondary antibody okay and it is basically bound with biotin so we call it as the biotinilated secondary

antibody so this is the particular antigen right so primary antibody is going to bind with the antigen in the

tissue and then that to the primary antibody if it has bound with the antigen then secondary biotin relateded

antibody is going to attach if they are specific right now now the avidin which is conjugated this is the avidin okay

this is the avidin this green is the avidin now this avidin conjugated with horse radish peroxidase this is the

horse radish or the enzyme this is the enzyme that is the horse radish peroxidase the avidin if you see it is

conjugated or it is attached with the peroxidase so if the primary antibody attaches with the antigen then only the

secondary biot antibody is going to attach with the prim primary antibody and this biotin present in the secondary

antibody will attach with the avidin peroxidized complex so once this avidin binds with the biotin so that particular

peroxidase is there so if I add a chromogen this peroxidase is going to have a reaction and give the color so

this is a basic concept so the biotinilated secondary antibody is tightly bound with the peroxidase

conjugated avidin so the advantage of this method is it is a rapid and sensitive test but what are the

disadvantages remember this is the tissue which is containing the antigen where we are detecting right but what

happens that the tissue may contain endogenous biotin which may react directly this aidin might directly react

with biotin present in the tissue so therefore a false positive reaction can happen secondly the Affinity of the

Biotin and avidin may vary widely in the different batches so the Affinity of the Biotin and avidin may vary widely in

different batches so they're not very stable so this may significantly affect the sensitivity and the reproducibility

of the test now comes the same avidin biotin test but now it is an aiin and biotin conjugated procedure now what

happens over here again the same thing when we had seen the indirect conjugated reaction and we had seen the peroxid

anti peroxid over here the same thing over here what we have done is that instead of using a single avidin over

here what we have have done that basically uh this biotin is there right and this is basically we are using this

particular avidin is this so what has happened that we have increased the number of avidin bound uh you know

biotin over here so what happens with a single antibody we are having multiple peroxidase action and as a result the

sensitivity of the test increases just in the similar manner that we are using H radish peroxidase antiperoxidase

system so over here also the same thing is happening so over here this is the particular antigen primary antibody the

secondary antibody that is the biotin lied is attaching over here and then it is attaching to particular avidin which

is already having a complex with different Biotin and which is attached with the peroxidase so as a result what

happens for a single antibody there is 1 2 three peroxidase so the reaction is very strong and there will be more color

so the reaction will detect even small amount of antigen thus the reaction will become highly sensitive so it is just a

modification of the above mentioned process to further increase the sensitivity of the test so here a

pre-formed complex of avidin Biotin and hos radish peroxides as we can appreciate over here okay is used now

the presence of multiple molecules of H radish peroxidase enhances the visualization process as I already told

you the steps are the same you use the primary antibody then you add the biotinated secondary antibody and then

you form you add the pre-formed complex of avidin Biotin and hos radish peroxidase it is a highly sensitive test

the disadvantage only again the same thing it can give false positive reaction because of the presence of

endogenous biotin in the tissues okay okay next now comes the biotin steav vidin method okay so over here we are

just replacing the avidin avidin is replaced with what is known as strepped avidin in this system the avidin is

replaced by a tetramic antibody Ste avidin that is directly conjugated with enzyme the stavin molecule is having a

very high Affinity against biotin the biotin stavin complex gives better amplification and detection as compared

to the avidin biotin complex okay so the advantage is that streptavidin does not cross react with lectin like substance

and the enzyme becomes more stable and can be stored for longer duration as this is directly bound with

streptavidin now the next important step that is the the next important iy reaction that is the alkaline

phosphatase anti- alkaline phosphatase method of I so over here it is very similar to the one that we had seen that

is with the hos radish peroxidase so you must be thinking if it is same why we are using this so let let let me tell

you so over here also the same thing is there there's an antigen now there's a primary antibody over here but over here

we are having a complex of alkaline phosphatase and anti- alkaline phosphatase so instead of peroxides anti

peroxides we are having this okay and in between they are bound by a secondary antibody which is highly specific both

for this alkaline phosphates anti- alkaline phosphates complex and which is also highly specific to the primary

antibody okay so basically what is happening over here in alkaline phosphotase anti- alkaline phas method

we use a complex of alkaline phosphatase anti- alkaline phosphatase as we can see over here this is that complex the

secondary antibody or linking antibody is used to bridge the primary antibody and this uh um up up complex now similar

to the peroxides anti peroxides method here also the primary antibody and antibody in apop complex they are from

same species whereas the secondary antibody that is a linking antibody is from different species now what is the

advantage of this and why we are using this instead of of the the usual peroxidase antiperoxidase method because

remember it is used in cases where the tissue contains high quantity of endogenous peroxidase activity such as

in case of bone marrow lymph node Etc so in bone marrow lymph node you cannot use the usual peroxidase anti peroxidase

method because itself these tissues are having endogenous peroxidase activity so they will give false positive reaction

also this is also used when we are doing dual imuno staining such as in a one slide you are using both the stain

different stains are used okay such as you using apop and peroxid anti peroxid also so in that case we are using both

of them now apop method provides a distinct bright red color which is easy to identify compared to the conventional

peroxid stain and apop is stable for a longer duration of time so I hope you understand what is the use of this

particular method now next important method is the polymer based labeling method so what is

the polymer based labeling method as you can appreciate in this technique a polymer backbone is used so this you can

appreciate this is a particular polymer backbone and nowadays we are most commonly using this only so this is a

particular polymer Backbone in this technique a polymer backbone is used so this polymer that we are seeing this can

be either a dextron a polypeptide or a dender polymer now the dextron polymer is often used as a polymer backbone now

what is seen over here as you can appreciate a large number of enzyme molecule at least you can see 100

peroxide a very large number of peroxide are are basically attached to this particular polymer backbone so a large

number of enzyme molecule at least 100 peroxides and more than 20 secondary antibodies more than 20 you can see this

is the secondary antibodies so over here more than 20 secondary antibodies are conjugated within this dextron polymer

as you can see so at first the primary antibody is applied followed by the dextron enzyme secondary antibody

complex okay this is the secondary antibody complex which is attached okay so what is the advantage of this method

it is simple and it is rapid two-step technique compared to the avidin biotin technique this is more sensitive why

more sensitive because you can appreciate over here there are multiple peroxidases so more number of enzymes so

they will catalyze more number of chromogen as a result more color will be formed so more sensitive because large

So This is highly sensitive because large number of enzymes are there which takes part and a single binding of

primary and secondary antibody and very important because over here there is no biotin involved so no background

staining as it bypasses the Biotin and avidin binding so there is no background staining as it bypasses the Biotin and

avidin bindings okay okay now the last and the most important and also the last and very important method that is the

catalyzed signal amplification that is the tyramine signal amplification method this is not regularly or routinely used

routinely this is the this hos radish peroxidase and uh this polymer based labeling method is routinely used

nowadays now in this method that is the catalyzed reporter deposition technique it is also known as tyramine signal

amplification method very easy it exploits the catalytic property of hos radish peroxidase in the presence of

hydrogen peroxide so biotinilated tyramine is used in presence of hos radish peroxidase and hydrogen peroxide

now the H radish peroxidase converts the biotinilated tyramine to a reactive biotinilated tyramide and this activated

biotinilated tyramide further reacts with tyrosine in the amino acid of tissues and deposits biotin so over here

we are depositing biotin inside the tissue the biotin this biotin is deposited only at the site of antigen

antibody reaction or at the site where the site of Interest this biotin is visualized by the avidin biotin

technique so let me show you with the help of a diagram then it's very easy to understand so over here we can see that

there is an act there there is basically this tyramide as you can appreciate okay and this tyramide this biotinilated

tyramine as you can appreciate over here biotinilated tyramine is there in presence of hos radish peroxidase and

H2O2 it is forming activated tyramide and this activated tyramide is acting with the tyrosine present in the tissues

okay and this is actually leading this reaction is leading to deposition of biotin in the reaction site okay as you

can appreciate over here okay and then via the normal avidin biotin Technique we are detecting this particular biotin

okay so over here what we are doing do initial hor radish so initially you make this H radish peroxides conjugated

system so as you can see this H radish peroxidase conjugate system is initially prepared okay then what happens you add

biotinated tarine solution to it so the biotinated uh tyramine solution is added and then you wash then you add the H

radish peroxidase conjugated stavin so whatever the hor radish peroxid conjugated stavin is ADD added and then

you have to add the dab to visualize the particular reaction over here okay so as you can see this is the basic your H

radish peroxidase over here as we can appreciate and to the hos radish peroxides we have added the biotinilated

tyramine as we can appreciate the activated biotinilated uh tyramine that is there that is the

amino acid that has been added over there okay and then you have added H radish peroxidase conjugated

streptavidin over here as you can see this is the end and then you add the chromosome to visualize the particular

reaction so let me just show you over here very you know smoothly once again so what has happened over here this is

the antigen we have added the primary antibody then the secondary antibody has been added now over here what we have

done first we have added the horse radish peroxidase we have added the horse radish peroxidase conjugated

system has been added after that whatever tyramine that is biotinilated tyramine okay which has already reacted

okay that is basically added and then we wash it so after washing all the UN wanted or

unreacted tyramine is going to go away and those which are bound these tyramine which are already bound over here as we

can appreciate okay so basically that is basically now binding with the hos radish peroxidase conjugated stavin is

added over here and then you add the chromogen to visualize the particular reaction so this method is a little bit

difficult method to understand okay so basically uh uh uh apart from this all these other methods are more important

okay so this polymer based labeling method is far more important then alkaline phosphates anti- alkaline

phosphates method is important biotin stavin method biotin aidin conjugated procedure aidin biotin simple method is

there then peroxid antiperoxidase method and then the direct and the indirect method so with this we have completed

the discussion of all the different types of methods which are employed okay in the IC process

okay so these are all the names of the techniques the primary second antibody the visualization system advantages and

the command so we have already discussed in details advantages disadvantages of all the methods I'm not repeating once

again you might go through this during the exam time so as to revise faster okay that is why included so now what is

the sample that you are using for imunohistochemistry as well as for immunocytochemistry so for

histopathology as I told ffp formal and fixed paraffin embedded tissue blocks are used and frozen section also but

nowadays it is not used and the use of ice in frozen section is highly discouraged then secondly it can be used

for cytology so for cytology if you're carrying out immunocytochemistry you have to use

cytology samples what are those as I told you cell block tissue smear from liquid based cytology direct cytology

smear the imprint or FY smear as well as the cytospin smear okay so these are the cytologic samples so how do you collect

the sample sample collection so histopathology ffp this is the most popular sample for ISC presently most of

the antibod is work on ffp tissue section frozen section the use is now discuss