Immunohistochemical staining (immunohistochemistry)

1.Immunohistochemical Staining's Principle

Immunohistochemistry is a technique for detecting peptides and proteins in tissues based on the principle of specific binding of antigens and antibodies. Now the following two detection methods are commonly used:
SP method:
Primary antibody + biotin-labeled secondary antibody + horseradish enzyme-labeled streptavidin + horseradish enzyme substrate color development

2. SABC Method of Immunohistochemical Staining:

Primary antibody + biotin-labeled secondary antibody + SABC (streptavidin + horseradish enzyme-labeled biotin) + horseradish enzyme substrate color development
Generally speaking, SP method has higher specificity and SABC method has higher sensitivity.

The final color reaction of immunohistochemistry is completed by the action of enzymes and substrates to generate insoluble pigments. The choice of substrate is closely related to the color reaction. The most commonly used is DAB, which is brownish yellow to tan precipitate.

3. Immunohistochemistry Process

1. Materials and reagents

2. Reagents: alcohol, xylene, citrate, PBS, primary antibody, secondary antibody kit, DAB, gum, etc.

3. Apparatus and equipment: slides, staining tank, wet box, pipette, microwave oven, incubator, refrigerator, microscope, etc.

4. Dyeing procedure

5. Dewax the slices to water; xylene I (15 minutes) → xylene II (15 minutes) → absolute ethanol I (5 minutes) absolute ethanol II (2 minutes) → 90% ethanol (2 minutes) → 80 % Ethanol (2 minutes) → 70% ethanol (2 minutes) → distilled water (2 minutes).

6. 3% H2O2 at room temperature for 10-20 minutes to inactivate endogenous enzymes. (30% H2O2 1 part + double distilled water 9 parts mixed to prepare fresh)
   Wash in PBS for 2 minutes × 2 times;

7. Microwave repair: Immerse the slice in 0.01M citrate buffer PH6.0) Electric stove or microwave oven is heated to boiling and then power off, after 2 to 3 minutes, repeat 2 to 3 minutes, tap water bath to room temperature, PBS Wash 2 minutes × 2 times;

8. Add normal goat serum blocking solution at room temperature for 20 minutes, without washing, shake off excess liquid. Block non-specific binding of tissues to antibodies and reduce background staining;

9. Drop Ⅰantibody (concentrate should be diluted in appropriate proportion, the amount of drop should be based on the size of tissue, 1cm×1cm size is about 40ul), 37℃ for 1-2 hours or 4℃ overnight; PBS wash 5 minutes × 3 times;

10. Add biotinylated secondary antibody dropwise, 37°C or room temperature for 30 minutes, wash with PBS for 5 minutes × 3 times;

11. Add the reagent complex dropwise, 37°C or room temperature for 30 minutes, wash with PBS for 5 minutes × 3 times;

12. DAB color development: use DAB color development kit, color development at room temperature, control reaction time under microscope, generally within 5 minutes;

13. Rinse with running water;

14. Hematoxylin counterstaining for 2 minutes;

15. Blue water flow for 15 minutes;

16. Roast dry in a drying oven, xylene transparent, neutral gum sealing slices, put into the oven baking slices.

4. Matters Needing Attention of Immunohistochemical Staining

1. The slice should be as thin, flat and free of knife marks as possible;

2. Before dropping reagents, shake off the sliced PBS first, and then wipe the water around the tissues with absorbent paper to prevent the loss of antibodies and dispersion, but the tablets cannot be dried.

Reagents related to immunohistochemistry:

Vibration section sealing solution (PNBS): (10ml)
 Final concentration: Stock solution:
 10% NS (Secondary Antibody Species Normal Animal Serum) NS 1ml
 2%BSA 10%BSA 2ml
 5% sucrose 20% sucrose 2.5ml
          +PBS PH7.4 to 10ml

MEMFA: (10ml)

Final concentration: Stock solution:
0.1M MOPS 1M MOPS 1ml
0.1mM EGTA 10mM EGTA 2ml
1mM MgSO4 1M MgSO4 10μl
3.7%PFA 10%PFA 3.7ml
+d2H2O to 10ml

AP-CDS: (100ml)

Final concentration: Stock solution:
 100mM Tris-Cl PH9.5 1MTris-Cl PH9.5 10ml
 50mM MgCl 1M MgCl 5ml
 100mM NaCl 5M NaCl 2ml
 0.1% Tween-20 Tween-20 100ml
 5mM levamisole 1M levamisole 500ml
+d2H2O to 100ml

HRP-CDS: (1ml)

(1) 6.6ml H2O2 in 100ml 0.1M TB PH 7.5
(2) 20ml DAB (0.05g/ml in d2H2O) + 5ml (1) solution + 0.1M TB PH 7.5 to 1ml

Preparation method of 10% paraformaldehyde: (100ml)

Weigh 10 grams of paraformaldehyde (SIGMA P6148) powder, place it in a 250ml Erlenmeyer flask, add 80ml 1xPBS, then add 5-6 drops (1ml pipette tip) of 10N NaOH, dissolve in a 65°C water bath for 3-4 hours After complete dissolution, cool to room temperature, adjust the pH value to 7.4, make the solution constant volume to 100ml, filter and divide into 4ml or 8ml per tube, and store at -20°C. Before use, dissolve the stock solution in an 80°C water bath, dilute it to 4% with 1xPBS, and use it. (After being prepared, store it in a sealed place at 4°C and use it within 24 hours.)
Or directly prepare 4% PFA, the method is the same as above (without adding NaOH to promote dissolution, there is no need to adjust the pH), after preparation, it is stored in a sealed place at 4°C and used within 24 hours.

0.5% gelatin processing slide method:

800ml treatment liquid:

1. Dissolve 4g gelatin and 4g chromium potassium sulfate in 800ml d2H2O, heat at 65°C for about 30min to dissolve (do not boil the solution)

2. Filter the solution and place the solution at room temperature to about 50°C.

3. Leach the slides 3-4 times in the solution.

4. Allow the treated slides to evaporate to dryness overnight at room temperature. To prevent dust, cover with plastic wrap.

5. Bake and dry for more than 3 hours at 130°C.

6. Store dry.
   After using the treatment solution, add 0.05% NaN3 and store at 4°C. Next time, heat the solution to 50°C.

Protein glue preparation method:

Take fresh egg whites, stir well, filter at 4°C, add equal volume of non-fluorescent glycerin to the clear solution, then add 0.05% NaN3 to mix thoroughly, divide into 1ml per tube, and store at -20°C.

Preparation method of fluorescent sealing tablet (Mowiol): (about 24ml)
Place 2.4g mowiol, 6g glycerin and 6ml deionized water in a 50ml centrifuge tube at room temperature for 2 hours until mowiol grows completely and becomes transparent. Add 12ml of 0.2M Tris buffer (pH 8.5) at 50°C (overnight) until the mowiol is completely dissolved. Centrifuge at 4000-5000 rpm for 20 minutes. Dispense into 1ml per tube and store at -20°C.

In diagnosis, histopathological diagnosis is considered as the gold standard to determine diseases and their natures. Before the body shows obvious clinical symptoms, the influence of drugs on the body tissues and organs can be found through histopathological examination, so as to provide reference for the prevention and treatment of diseases. In drug filing, all relevant institutions pay great attention to the results of histopathological diagnosis because such results can provide important basis for judging the potential harm degree of the drugs to be filed.

The guidelines issued by various regulatory agencies also require histopathological diagnosis results of short-term toxicity, subchronic toxicity, chronic toxicity and carcinogenicity tests. As a comprehensive CRO enterprise, Medicilon provides considerate services in histopathology.

5. Medicilon's Immunohistochemical Staining Services: 

 -In the aspect of pathological diagnosis, we can provide pathological diagnosis services for many kinds of animals, including but not limited to rats, mice, guinea pigs, rabbits, dogs, non-human primates and miniature pigs; 

-In terms of toxicity tests, we are proud of our rich experience, especially in short-term toxicity tests, subchronic toxicity tests, chronic toxicity tests and carcinogenicity research; 

-As for diagnosis requirements of special tissues and organs put forward by some clients, we can provide active exploration methods and solutions to fully meet the pathological diagnosis requirements including drug filing. 

-In terms of different staining techniques, in addition to routine paraffin section H&E staining, we are also available for frozen section, special staining, immunohistochemical staining, TCR, etc. to provide diagnostic services, so as to meet the needs of clients for tissue observation of specific tests.