Immunohistochemistry Protocol

Product Manager: Harrison Michael

I. Immunohistochemistry - Protocol for Paraffin Sections

Important Note: Please refer to the application section on the front page of the product data sheet or product webpage on Aladdin's official website to confirm whether the product is suitable for paraffin-embedded (IHC-P) tissue sections.

Note: For appropriate antibody dilution, diluents, and unmasking solutions, please refer to the product-specific protocol.

A. Solutions and Reagents

Note: Prepare solutions using reverse osmosis deionized water (RODI) or water of equivalent grade.

1. Xylene. X112051

2. Anhydrous denatured ethanol, tissue grade (100% and 95%). E298965

3. Deionized water (dH₂O). W119424

4. Washing buffer:

a. 1× Tris Buffered Saline with Tween 20 (TBST): To prepare 1 L of 1× TBST, add 100 mL of 10× Tris Buffered Saline with Tween 20 (TBST) (T743331) to 900 mL of dH₂O and mix well.

5. *Choice of antibody diluents:

a. TBST/5% Normal Goat Serum: Add 250 µL of Normal Goat Serum (G752123) to 5 mL of 1× TBST.

b. PBST/5% Normal Goat Serum: Add 250 µL of Normal Goat Serum (G752123) to 5 mL of 1× PBST.

• 1× PBST: To prepare 1 L of 1× PBST, add 100 mL of 10× Phosphate Buffered Saline with Tween 20 (PBST) (P196391) to 900 mL of dH₂O and mix well.

6. Choice of antigen retrieval reagents:

a. 1× Citrate Retrieval Solution (C752119): To prepare 250 mL of citrate retrieval solution, dilute this antigen retrieval solution (50×) 50-fold with double-distilled water or Milli-Q water to prepare 1× antigen retrieval solution. For example, add 5 mL of this antigen retrieval solution (50×) to 245 mL of double-distilled water or Milli-Q water, mix well to obtain 250 mL of 1× antigen retrieval solution.

b. 1× EDTA Retrieval Solution E745530: To prepare 250 mL of 1× EDTA retrieval solution, dilute 5 mL of EDTA antigen retrieval solution (50×) (E745530) with 245 mL of dH₂O.

c. TE: 10 mM Tris/1 mM EDTA, pH 9.0: To prepare 1 L of solution, add 1.21 g of Tris base (C₄H₁₁NO₃) T110601 and 0.372 g of EDTA (C₁₀H₁₄N₂O₈Na₂•2H₂O) E274376 to 950 mL of dH₂O. Adjust the pH to 9.0, then bring the final volume to 1 L with dH₂O.

d. Trypsin T105533 or Pepsin P736658: Pre-warm to 37°C before use; digestion time is approximately 5-30 minutes.

7. 3% Hydrogen Peroxide H639762: To prepare 100 mL of solution, add 10 mL of 30% H₂O₂ to 90 mL of dH₂O.

8. Blocking solution: 1× TBST/5% Normal Goat Serum or 1× Animal-Free Blocking Solution.

a. 1× TBST/5% Normal Goat Serum: Add 250 µL of Normal Goat Serum to 5 mL of 1× TBST.

b. Immunostaining Blocking Solution: B751639.

9. Detection systems: HRP, Mouse Ab179001; HRP, Rabbit Ab176443; Biotin, Mouse Ab156253; Biotin, Rabbit Ab176442; HRP-conjugated Streptavidin np156148

10. DAB Staining Kit: Detection Reagent D405772

11. Hematoxylin: Hematoxylin H104304

12. Mounting Medium: Mounting Medium M292692

B. Deparaffinization/Rehydration

Note: Ensure that sections remain moist throughout the procedure.

1. Deparaffinization/hydration steps:

a. Incubate sections in xylene washes 3 times, 5 minutes each.

b. Incubate sections in 100% ethanol 2 times, 10 minutes each.

c. Incubate sections in 95% ethanol 2 times, 10 minutes each.

d. Wash sections with dH₂O twice, 5 minutes each.

C. Antigen Retrieval

Note: Refer to the product-specific protocol for specific recommendations on retrieval solutions/procedures.

1. For citrate: Immerse sections in 1× citrate retrieval solution, then heat in a microwave until boiling; maintain sub-boiling temperature (95°C-98°C) for 10 minutes. Cool sections on the bench for 30 minutes.

2. For EDTA: Immerse sections in 1× EDTA retrieval solution, then heat in a microwave until boiling; maintain sub-boiling temperature (95°C-98°C) for 15 minutes. No cooling required.

3. For TE: Immerse sections in 10 mM Tris/1 mM EDTA solution (pH 9.0), then heat in a microwave until boiling; maintain sub-boiling temperature (95°C-98°C) for 18 minutes. Cool sections on the bench for 30 minutes.

4. For trypsin or pepsin: Pre-warm to 37°C before use; digestion time is approximately 5-30 minutes.

D. Staining

Note: Refer to the product-specific protocol for recommended antibody diluents.

1. Wash sections with dH₂O three times, 5 minutes each.

2. Incubate sections in 3% hydrogen peroxide aqueous solution for 10 minutes.

3. Wash sections with dH₂O twice, 5 minutes each.

4. Wash sections with washing buffer for 5 minutes.

5. Apply 100–400 µL of preferred blocking solution to each section, block at room temperature for 1 hour.

6. Remove blocking solution, then add 100–400 µL of primary antibody diluted in the recommended antibody diluent to each section.

7. Incubate at 4°C overnight.

8. Equilibrate Detection Reagent to room temperature.

9. Remove antibody solution, wash sections with washing buffer 3 times, 5 minutes each.

10. Apply appropriate amount of secondary antibody to cover sections as needed. Incubate at room temperature in a humidified chamber for 30 minutes.

11. Wash sections with washing buffer 3 times, 5 minutes each.

12. Prepare substrate as recommended in the product usage information.

13. Apply 100-400 µL of DAB to slides. Monitor the reaction. Recommended reaction times vary by substrate used. Refer to product usage information for specific guidelines.

14. Immerse sections in dH₂O.

15. If needed, counterstain sections with Hematoxylin according to instructions.

16. Wash sections with dH₂O twice, 5 minutes each.

17. Dehydrate sections:

 a. Incubate sections in 95% ethanol 2 times, 10 seconds each.

 b. Repeat in 100% ethanol 2 times, 10 seconds each.

 c. Repeat in xylene 2 times, 10 seconds each.

18. Mount with coverslip and mounting medium.

II. Immunohistochemistry Protocol - For Frozen Sections

Important Note: Refer to the product webpage to confirm if a product is validated and approved for frozen tissue sections. See the product webpage for appropriate antibody dilutions and retrieval solutions.

Note: See the product webpage for product-specific protocol recommendations.

A. Solutions and Reagents

Note: Prepare solutions using reverse osmosis deionized water (RODI) or water of equivalent grade.

1. Xylene. X112051

2. Anhydrous denatured ethanol, tissue grade (100% and 95%). E298965

3. Deionized water (dH₂O). W119424

4. Hematoxylin (optional): Hematoxylin H104304.

5. 20× Phosphate Buffered Saline (PBS): P492453 To prepare 1 L of 1× PBS: Add 50 mL of 20× PBS to 950 mL of dH₂O, then mix well.

6. Choice of fixatives: Refer to the product data sheet for the optimal fixative.

 a. 10% Neutral Buffered Formalin. F301880

 b. Acetone. A399740

 c. Methanol. M116124

 d. 3% Formaldehyde: To prepare 100 mL of solution, add 18.75 mL of 16% formaldehyde to 81.25 mL of 1× PBS.

7. 10× Tris Buffered Saline (TBS) washing buffer: (T743331) To prepare 1 L of 1× TBS solution, add 100 mL of 10× TBS to 900 mL dH₂O and mix well.

8. Methanol/Peroxidase: Preparation method: Add 10 mL of 30% H₂O₂ to 90 mL of methanol. Store at -20°C.

9. Blocking solution: 1× TBS/0.3% Triton X-100/5% Normal Goat Serum. Preparation method: Add 500 µL of goat serum and 30 µL of Triton X-100 to 9.5 mL of 1× TBS.

10. Detection systems: HRP, Mouse Ab179001; HRP, Rabbit Ab176443; HRP, Goat Ab141534;

11. DAB Staining Kit: Detection Reagent D405772

B. Section Preparation

1. For tissues stored at -80°C: Before sectioning, remove tissue samples from the freezer and equilibrate at -20°C for approximately 15 minutes. This prevents sections from cracking during blocking.

2. Cut tissues into sections with a thickness of 6–8 µm and place on positively charged slides.

3. Before fixation, air-dry sections on the bench for a few minutes (this enhances section adhesion).

C. Choice of Fixatives

Note: Refer to the product data sheet to determine the optimal fixative.

1. After tissue sections are air-dried, fix with the optimal fixative according to the following steps.

a. 10% Neutral Buffered Formalin: Fix at room temperature for 10 minutes. Proceed immediately to staining (Section D).

b. Frozen acetone: Freeze at -20°C for 10 minutes. Air-dry. Proceed immediately to staining (Section D).

c. Methanol: Freeze at -20°C for 10 minutes. Proceed immediately to staining (Section D).

d. 3% Formaldehyde: Fix at room temperature for 15 minutes. Proceed immediately to staining (Section D).

e. 3% Formaldehyde/methanol: Fix in 3% formaldehyde solution at room temperature for 15 minutes, then fix in methanol at -20°C for 5 minutes (do not rinse during transfer). Proceed immediately to staining (Section D).

D. Staining

1. Wash sections with washing buffer twice for 5 minutes.

2. Incubate in methanol/peroxidase at room temperature for 10 minutes.

3. Wash sections with washing buffer twice for 5 minutes.

4. Apply 100–400 µL of blocking solution to each section, block at room temperature for 1 hour.

5. Remove blocking solution, then add 100–400 µL of primary antibody diluted in blocking solution to each section.

6. Incubate at 4°C overnight.

7. Equilibrate DAB to room temperature.

8. Remove antibody solution, wash sections in washing buffer 3 times, 5 minutes each.

9. Apply appropriate amount of secondary antibody to cover sections as needed. Incubate at room temperature in a humidified chamber for 30 minutes.

10. Wash sections with washing buffer 3 times, 5 minutes each.

11. Apply 100–400 µL of DAB to each section and observe closely; appropriate staining intensity is usually achieved within 1–10 minutes.

12. Immerse sections in dH₂O.

13. If needed, counterstain sections with Hematoxylin according to the manufacturer's instructions.

14. Wash sections with dH₂O twice, 5 minutes each.

15. Dehydrate sections:

 a. Incubate sections in 95% ethanol 2 times, 10 seconds each.

 b. Repeat in 100% ethanol 2 times, 10 seconds each.

 c. Repeat in xylene 2 times, 10 seconds each.

 d. Cover sections with coverslips.

Aladdin：https://www.aladdinsci.com/