Immmunohistochemical (IHC) Methods: Immunoperoxidase Staining

Immunoperoxidase Staining

1. Basics
2. The Theory of the ABC Technique
3. Fixation
4. Advantages
   1. Immunoperoxidase Procedure
   2. ABC Method
5. Summary of Steps in ABC Procedure
6. Cutting
7. Dilution of Antibodies by Checkerboard Titration
8. Antisera & Related Reagents of ABC Method
9. Primary Antisera Chart
10. Working Solutions
11. Procedure
12. Result
13. Discussion

Basics

There are TWO MAIN GOALS in the immunoperoxidase technique. The first is to seek out and identify a specific antigen in tissue. This is accomplished by developing an antibody with high affinity for the antigen in question and then incubating it with the tissue where it will bind chemically to that antigen. The second major goal is to make the antibody-antigen complex visible. This is accomplished by marking the site with dye molecules which are discernible under a standard light microscope.

The Theory Of The Abc Technique

The immunoperoxidase technique used in the student labs is the ABC technique or the avidin-biotin complex technique. This procedure employs primary antibody, biotinylated secondary antibody, and a pre-formed Avidin: Biotinylated horseradish peroxidase Complex (ABC). Avidin, an egg white protein (molecular weight – 68,000), has an extraordinary affinity for the naturally occurring small molecule vitamin biotin (dissociation constant 10-15). The conjugation of biotin to antibodies or peroxidase molecules enables them to bind avidin molecules. Avidin has four binding sites for biotin molecules, and biotin-labeled peroxidase can be prepared as a form of one peroxidase molecule containing several biotin molecules. Therefore, during the formation of ABC, avidin acts as a bridge between biotin-peroxidase molecules, and biotin-peroxidase serves as a link between avidin molecules. It is likely that a lattice-like complex, containing several peroxidase molecules, is formed. Since an excess of avidin is added, the biotin-bonding sites in the complex are not completely occupied by biotin molecules associated with the secondary antibody, giving a superior sensitivity.

Fixation

Immunoperoxidase methods have been reported to be successful when used in conjunction with a wide variety of fixatives. This has been one of the major benefits of these methods. Bouin’s fluid, formalin, Zenker’s, B5 and glutaraldehyde have been used in various immunocytochemical procedures. However, high concentrations of fixatives as well as prolonged exposure to any given fixation will destroy antigens. It is advantageous to fix for the shortest possible time that is consistent with excellent morphologic detail. Autopsy tissue or tissues that have begun to autolyze have proven to be virtually unusable for immunohistochemistry. If formalin is used, it should be fresh and buffered to prevent drastic

changes to the antigens. Always check the pH and discard if a white precipitate forms. When using Zenker’s or B5 fixative, be sure to remove mercury pigment before running any immunoperoxidase technique. If Bouin’s fixative is used, be sure adequate washing has been performed to remove picric acid. Cytological preparations may be fixed in acetone for 10 minutes (ie. B.M) and then allowed to air dry.

Advantages

Immunoperoxidase Procedures

Immunoperoxidase is one means to provide a distinct stain at the antigen-antibody sites. Some advantages of the immunoperoxidase procedure over other methods are as follows:

1. The stain permits the pathologist to simultaneously evaluate the morphology (structure) of a cell or tissue section using traditional criteria. The possibility of diagnosing a difficult case is greatly enhanced when knowledge of specific antigen sites is added to the morphological information.
2. The antigen-antibody complex can be visualized within minutes by using an ordinary light microscope.
3. Immunoperoxidase staining can be performed on tissues that have been routinely preserved (fixed) and stored (paraffin embedded) by hospital laboratories. This allows the pathologist to do retrospective studies as well as current evaluations.
4. The labeling (staining) of antigen sites is permanent and the cell or tissue sections can be stored for future reference.
5. The immunoperoxidase method permits investigators to study functional aspects of many tumors. For example, does the distribution of cellular components change during malignant (cancerous) transformation? Studies like these can result in better understanding of normal and abnormal cell types.

ABC Method

There are a number of advantages of the ABC method as compared to other peroxidase techniques:

1. The ABC method is approximately eight to forty times more sensitive than the PAP (peroxidase-anti-peroxidase method).
2. It has a low level of non-specific background staining.
3. In this method, a higher dilution of primary antibody can be used (therefore cheaper).
4. As well as a higher dilution of the secondary antibody, the use of low concentration of antibodies minimizes background, increases method specificity and reduces cost for antibodies.
5. The ABC reagents can be used for all systems and are contingent only upon a biotinylated antibody.
6. Any substance tagged with biotin can be detected by the ABC techniques. Therefore, the method is quite flexible and has a wide range of research applications.

Summary Of Steps In Abc Procedure

1. The section is treated with hydrogen peroxide, an endogenous peroxidase blocking reagent, which destroys any natural peroxidase activity that may be present, e.g. RBC and many WBC.
2. Incubate sections with normal sera to suppress non-specific binding of immunoglobulins to specimens, particularly those rich in collagen.
3. Apply “Primary Antibody” – the antibody against the antigen to be demonstrated. It will bind to the antigen sites on the section.
4. After a rinse with PBS to remove unbound primary antibody, a “Secondary Antibody” is added which has been “biotin” labeled. This serves as a “link” in the ABC technique by introducing biotin into the section at the location of the primary antibody.
5. Section is again washed with PBS and the avidin: biotinylated horseradish peroxidase macromolecular complex (ABC) is then added. This binds to the biotinylated secondary antibody.
6. Unbound ABC reagent is then removed by washing with PBS. Finally, a substrate solution consisting of hydrogen peroxide and DAB (Sigma 3′, 3′ -diamino-benzidine) is added. In a reaction catalyzed by peroxidase, the DAB is oxidized to form a dark brown color at the antigen sites.
7. Hematoxylin is used as the counterstain for nuclear detail and to enhance microscopic resolution.

Cutting

Sections are cut at routine thickness around 4-5 µm. Mount sections on “charged” slides. Etch around section with a diamond marker so solution won’t run off. Two slides are needed per stain per block (one serves as a test slide, the other serves as a negative control).

DILUTION OF ANTIBODIES

In the ABC technique, two antibody solutions are used. The optimal dilution is determined for each antibody. The ABC complex is purchased from Vector Laboratories and the recommended dilution of avidin and biotinylated horseradish peroxidase to PBS will be used.

Antisera & Related Reagents For ABC Method

Antibodies, which are less stable, are divided into small aliquots upon arrival and kept at -70°C. Others are kept in 4°C in refrigerator. Dilution should be done just before use.

Primary Antisera Chart

* H: high temperature antigen unmasking technique for paraffin sections using citrate buffer (pH 6.0)

Working Solutions

1. Xylene
2. Absolute ethanol, 95% ethanol, 70% ethanol.
3. Endogenous peroxidase blocking reagent
   

   100% Methanol	25 mL
   3% Hydrogen Peroxide	25 mL

(3% hydrogen peroxide used here should be prepared fresh each week from 30% H2O2 stock) Store in the refrigerator.

4. Suppressor Serum

The most effective suppressor serum for this method is a normal serum from the animal where the secondary antibody is produced. For example if the secondary antibody is from horse, then we use normal horse as suppressor serum.

Working dilution is 1/20. (i.e. Normal serum-1 part, PBS-19 parts)

Quantity required: 0.5 mls is required per slide approximately. i.e. One tube holds 4 mLs of PBS; add 200 µl normal horse sera (pre-measured in the microcup and frozen in the -70OC ) with an Eppendorf pipette, rinse the tip and mix well with a glass Pasteur pipette.

5. Phosphate Buffered Saline (PBS) Ph 7.2 Stock Solution

Stock solution:

OR

Make up 2 liters with distilled water.

Adjust pH to 7.2 by adding 40% Na0H drop by drop.

We purchase

Working solution of phosphate buffer:

Dilute stock solution 1/10 with distilled water and put into clean-labeled “squirt” bottle.

6. Primary Antibody

Most are anti-human antibody produced in rabbit. The monoclonal antibody for B cell (CD-20) is made in mouse. Refer to table for optimal dilution in section “I”. This reagent is very expensive and should not be wasted. Time should be taken to accurately assess the amount of working antibody needed. Label each tube with antibody and dilution.

B cell (CD-20): – 0.02 ml (20µl) aliquot frozen at – 70°C

• 1/100 dilution is needed
• pipette 2 ml of working PBS into a tube; use a long tip glass Pasteur pipette to transfer the 20µl of antibody into the tube with PBS, rinse the Eppendorf pipette tip and the microcup with PBS and then mix the dilution with a glass Pasteur pipette

7. Secondary Antibody (Linking)

When choosing the secondary antibody to use, choose the biotinylated antibody against the animal species where primary antibody is produced. For example, if the primary antibody is from mouse, then we use biotinylated anti-mouse immunoglobulin fraction.

Biotinylated anti-mouse IgG (from horse) for the CD-20

Working dilution is 1:200

Example – 0.02 ml (20µl) aliquot frozen at – 70°C

• pipette 4 mLs of PBS in tube
• use a long tip glass Pasteur pipette to transfer the 20µl of antibody into the tube with PBS, rinse the Eppendorf pipette tip and the microcup with PBS and then mix the dilution with a glass Pasteur pipette

8. ABC (Avidin-Biotin Complex Reagent)

Prepare 1/2 hr. before use to allow the complex to form.

Dilution

• add 10.0 mLs of PBS to the special working container provided in the kit
• add 0.1 mL or 100 µl reagent A (avidin) from the “A” microcup, mixing well.
• then add 0.1 mL or 100 µl reagent B (biotinylated peroxidase) from the “B” microcup, mixing well.
• allow to set for at least 30 minutes, but is stable for 72 hours if stored in the refrigerator

9. Chromogen/Developer Solution (DAB)

This solution should be prepared immediately before use. Care should be taken to avoid inhalation of the powder or contact with the skin (carcinogen). WEAR GLOVES AND MIX UNDER AN ENCLOSED FUMEHOOD. Each pair of students can make up one container of chromogen.

1. Pre-measure 25 mLs of PBS in an empty covered urine container.
2. With 2 minutes remaining in step 18, remove the DAB tablet from the -20OC freezer and add to the PBS. [10 mg of DBA (Sigma 3′, 3′-Diamino-benzidine)]. The DAB should dissolve immediately (you may “swirl” slightly but be careful not to contaminate the lid of the container)
3. With 2 minutes remaining in step 19, add 0.01 mL or 10 µl of concentrated (30%) hydrogen peroxide to this DAB solution.

Working Solutions

1. Xylene
2. Absolute ethanol, 95% ethanol, 70% ethanol.
3. Endogenous peroxidase blocking reagent
   

   100% Methanol	25 mL
   3% Hydrogen Peroxide	25 mL

(3% hydrogen peroxide used here should be prepared fresh each week from 30% H2O2 stock) Store in the refrigerator.

4. Suppressor Serum

The most effective suppressor serum for this method is a normal serum from the animal where the secondary antibody is produced. For example if the secondary antibody is from horse, then we use normal horse as suppressor serum.

Working dilution is 1/20. (i.e. Normal serum-1 part, PBS-19 parts)

Quantity required: 0.5 mls is required per slide approximately. i.e. One tube holds 4 mLs of PBS; add 200 µl normal horse sera (pre-measured in the microcup and frozen in the -70°C ) with an Eppendorf pipette, rinse the tip and mix well with a glass Pasteur pipette.

5. Phosphate Buffered Saline (PBS) Ph 7.2 Stock Solution

Stock solution:

OR

Make up 2 liters with distilled water.

Adjust pH to 7.2 by adding 40% Na0H drop by drop.

We purchase

Working solution of phosphate buffer

Dilute stock solution 1/10 with distilled water and put into clean-labeled “squirt” bottle.

6. Primary Antibody

Most are anti-human antibody produced in rabbit. The monoclonal antibody for B cell (CD-20) is made in mouse. Refer to table for optimal dilution in section “I”. This reagent is very expensive and should not be wasted. Time should be taken to accurately assess the amount of working antibody needed. Label each tube with antibody and dilution.

B cell (CD-20): – 0.02 ml (20µl) aliquot frozen at – 70°C

– 1/100 dilution is needed

– pipette 2 ml of working PBS into a tube; use a long tip glass Pasteur pipette to transfer the 20µl of antibody into the tube with PBS, rinse the Eppendorf pipette tip and the microcup with PBS and then mix the dilution with a glass Pasteur pipette

7. Secondary Antibody (Linking)

When choosing the secondary antibody to use, choose the biotinylated antibody against the animal species where primary antibody is produced. For example, if the primary antibody is from mouse, then we use biotinylated anti-mouse immunoglobulin fraction.

Biotinylated anti-mouse IgG (from horse) for the CD-20

Working dilution is 1:200

Example – 0.02 ml (20µl) aliquot frozen at – 70°C

• pipette 4 mLs of PBS in tube
• use a long tip glass Pasteur pipette to transfer the 20µl of antibody into the tube with PBS, rinse the Eppendorf pipette tip and the microcup with PBS and then mix the dilution with a glass Pasteur pipette

8. ABC (Avidin-Biotin Complex Reagent)

Prepare 1/2 hr. before use to allow the complex to form.

Dilution

• add 10.0 mLs of PBS to the special working container provided in the kit
• add 0.1 mL or 100 µl reagent A (avidin) from the “A” microcup, mixing well.
• then add 0.1 mL or 100 µl reagent B (biotinylated peroxidase) from the “B” microcup, mixing well.
• allow to set for at least 30 minutes, but is stable for 72 hours if stored in the refrigerator

9. Chromogen/Developer Solution (DAB)

This solution should be prepared immediately before use. Care should be taken to avoid inhalation of the powder or contact with the skin (carcinogen). WEAR GLOVES AND MIX UNDER AN ENCLOSED FUMEHOOD. Each pair of students can make up one container of chromogen.

1. Pre-measure 25 mLs of PBS in an empty covered urine container.
2. With 2 minutes remaining in step 18, remove the DAB tablet from the -20OC freezer and add to the PBS. [10 mg of DBA (Sigma 3′, 3′-Diamino-benzidine)]. The DAB should dissolve immediately (you may “swirl” slightly but be careful not to contaminate the lid of the container)
3. With 2 minutes remaining in step 19, add 0.01 mL or 10 µl of concentrated (30%) hydrogen peroxide to this DAB solution.

PROCEDURE

Before beginning the staining process, the slides should be labeled with the antigen to be localized. One slide from each block is needed per test antigen. One slide from each block serves as a negative control by omitting the primary antibody. A known positive control slide should be run with each test.

Students will only run a positive and negative control.

Notes

• Working solution of phosphate buffer: Dilute stock solution (purchased) 1/10 with distilled water and put into clean labeled “squirt” bottle.
• A “well” must be made around the tissue to contain reagents. Etch around the section with diamond marker.

1. Deparaffinize well in xylene. Rehydrate through graded alcohols to water.
2. Microwave Retrieval Of Antigen (Use for BCell Method)
   1. Dewax and rehydrate section.
   2. Rinse in distilled water.
   3. Place sections in plastic coplin jar containing 50 mL of citrate buffer. Cover jar loosely.
   4. Place jar in the middle of the microwave oven. Heat sections 2 x 1min.
   5. Leave sections in jar at Room Temperature for 20 minutes.
   6. Rinse in Distilled water and proceed with IHC staining.
3. Place slides in freshly prepared endogenous peroxidase blocking reagent (make 1/group) for 30 min. in a covered coplin jar.

Endogenous peroxidase blocking reagent

(3% hydrogen peroxide used here should be prepared fresh each week from 30% H2O2 stock). Store in the refrigerator.

4. Wash in running tap water 5 min.
5. Place slides in the moisture chamber and flood with PBS for 5 minutes.
6. Drain and flood with PBS for 5 minutes.
7. Remove slide from chamber, drain and carefully wipe away excess liquid from around the section using gauze.
8. Incubate in moisture chamber with background suppressor serum (make 1/group) for 30 minutes. Make up primary dilution in last 5 minutes of incubation (see step #9) Background Suppressor Serum: Add 200 µl normal horse sera (secondary antibody is anti-mouse prepared in a horse) with an Eppendorf pipette, rinse the tip and mix well with a glass Pasteur pipette. This will provide enough suppressor for 4 slides.
9. Tap off serum and wipe away excess. It is not necessary to wash with PBS at this time. Apply primary antisera (anti- CD 20 or B cell produced in a mouse) and incubate for 30 minutes. Apply PBS to the negative control.Primary Antibody Dilution for B cell (CD-20): 0.02 ml (20µl) aliquot frozen at – 70°C1/100 dilution is needed. Pipette 2 ml of working PBS into a tube; use a long tip glass Pasteur pipette to transfer the 20µl of antibody into the tube with PBS, rinse the pipette tip and the microcup with PBS and then mix the dilution with a glass Pasteur pipette. Provides enough for 2 slides. Each student should make this dilution
10. Squirt wash with PBS, drain and then immerse in PBS for 5 min.
11. Squirt wash with PBS, drain and then immerse in PBS for 5 min. Make up secondary antibody dilution (see step #12).May be held at this point in the fridge overnight in a tupperware container with brown paper saturated with distilled water on the bottom to maintain a moist environment.If in fridge, rinse one more time with PBS (as above) before proceeding.
12. Remove slide from chamber and carefully wipe away excess liquid from around the section.Apply secondary biotinylated antibody (anti-mouse from a horse) and incubate for 30 minutes. Prepare ABC reagent, as it requires ½ hour before use for the complex to form. (see step #15)Biotinylated anti-mouse IgG (from horse) for the CD-20:0.02ml (20µl) aliquot frozen at – 70°C1:200 dilution is needed. Pipette 4 mLs of PBS into a tube; use a long tip glass Pasteur pipette to transfer the 20µl of antibody into the tube with PBS, rinse the Eppendorf pipette tip and the microcup with PBS and then mix the dilution with a glass Pasteur pipette Provides enough for 4 slides (0.5 mLs approximately each slide). Make up 1/group
13. Squirt wash with PBS, drain and then immerse in PBS for 5 min.
14. Squirt wash with PBS, drain and then immerse in PBS for 5 min.
15. Remove slide from chamber and carefully wipe away excess liquid from around the section.Apply ABC reagent and incubate for 30 minutes. ABC (Avidin-Biotin Complex Reagent): Prepare 1/2 hr. before use to allow the complex to form. Dilution: Add 10.0 mLs of PBS to the special working container provided in the kit. Add 0.1 mL or 100 µl reagent A (avidin) from the “A” microcup, mixing well then add 0.1 mL or 100 µl reagent B (biotinylated peroxidase) from the “B” microcup, mixing well. (use Eppendorf pipette) Allow to set for at least 30 minutes, but is stable for 72 hours if stored in the refrigerator. Make up 3/lab group
16. Squirt wash with PBS, drain and then immerse in PBS for 5 min.
17. Squirt wash with PBS, drain and then immerse in PBS for 5 min.
18. Transfer the slides in the humidity chamber to the fume hood. Remove slide from chamber and carefully wipe away excess liquid from around the section. Slides should now be transferred to a staining rack in the “DAB” plastic container in the fumehood, as the chromogen (DAB) is carcinogenic. Chromogen/Developer Solution (DAB): This solution should be prepared immediately before use. Care should be taken to avoid inhalation of the powder or contact with the skin (carcinogen). WEAR GLOVES AND MIX UNDER AN ENCLOSED FUMEHOOD. Each pair of students can make up one container of chromogen.
    1. Pre-measure 25 mLs of PBS in an empty covered urine container.
    2. With 2 minutes remaining in step # 16, remove the DAB tablet from the -20OC freezer and add to the PBS. [10 mg of DBA (Sigma 3′, 3′-Diamino-benzidine)]. The DAB should dissolve immediately (you may “swirl” slightly but be careful not to contaminate the lid of the container)
    3. With 2 minutes remaining in step # 17, add 0.01 mL or 10 µl of concentrated (30%) hydrogen peroxide to this DAB solution.
    4. Make 1 container of DAB/group.All DAB residue and the forceps used for DAB should remain in this container in the hood. Apply chromogen solution for 2 minutes using a Pasteur pipette. Transfer the slides in distilled water back to the sink in histology and wash well in tap water, counter stain in Mayer’s hematoxylin for 2 min. Wash in running tap water. Blue in lithium carbonate for 1 minute (do not agitate as sections could fall off slide in alkaline environment) or until sections are “blue”.
19. Wash in water, dehydrate, clear in xylene and mount.

Result

When DAB is used as the chromogen, the positive tissue antigen sites are demonstrated in dark brown color. Nuclei are blue.

Discussion

1. Enzyme Digestion – It was generally believed that aldehyde fixation might produce chemical changes causing partial or total loss, destruction, or inaccessibility of the tissue antigenic sites. The treatment of tissue sections with proteolytic enzyme before routine immunohistochemical staining was found to be helpful in some cases. One of the most commonly used proteolytic enzymes is Trypsin.
2. Microwave Retrieval Of Antigen (Use for BCell Method)
   1. Dewax and rehydrate section.
   2. Rinse in distilled water.
   3. Place sections in plastic coplin jar containing 50 mL of citrate buffer. Cover jar loosely.
   4. Place jar in the middle of the microwave oven. Heat sections 2 x 1min.
   5. Leave sections in jar at Room Temperature for 20 minutes.
   6. Rinse in Distilled water and proceed with IHC staining.
      1. 0.01M CITRATE BUFFER
         1. Citric acid anhydrate 1.92g
         2. Distilled Water 900mL
      2. Adjust pH to 6.0 with approx. 13 mL of 2M NaOH. Make to final volume of 1000 mL with Distilled Water. Check pH.
         1. 2M NaOH: NaOH 8g
         2. Distilled Water 100mL
3. High Ph Buffer Antigen Retrieval
   1. Fill waterbath with hot water 2/3 full. Plug in and let water warm up beyond 90°C.
   2. Fill a microwavable coplin jar with 50 mL of HIGH pH TARGET ANTIGEN RETRIEVAL SOLUTION; cover jar loosely.
   3. Place jar in the middle of the microwave oven. Microwave at high power for 1 minute, 30 seconds.
   4. Immerse room temperature deparaffinized and rehydrated sections into preheated target retreival solution. Incubate for 30 min in hot water bath.
   5. Remove coplin jar from water bath and let sit at room temperature for an additional 20 minutes.
   6. Rinse in Distilled water and proceed with IHC staining.
4. Steamer Method for Antigen Retrieval
   1. Fill steamer with Distilled water to max water level fill line.
   2. Place plastic coplin jars containing 50mL working citrate buffer Steamer Method for Antigen Retrieval (ph 6.0) for antigen retrieval in vegetable steamer and turn on for 20 min.
   3. Place slides in pre-warmed citrate buffer in coplin jars. .
   4. Leave in steam for 30 more min at full steam.
   5. Let cool for 20 min
   6. Rinse in Distilled water and proceed with IHC staining.
5. Post Fixation
   1. Formalin-fixed, paraffin embedded tissue sections could be post fixed in various fixatives to enhance staining. Bring sections down to water. Post fix section in fixative of choice (e.g. B5, Bouin’s, etc.) for 1 hr. Remove mercury pigments if needed and continue with ABC staining procedure (step 3) to finish.

References

IWK. (2007). IWK Laboratory Histology Staining Manual, Special Stains.