Immunohistochemical Staining of S-100 Protein in Human Rectal Suction Biopsy Sections

Abstract

Source: Chi, S., Fang, et al. Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5. J. Vis. Exp. (2019)

This video demonstrates the immunohistochemical staining for S-100 protein in rectal suction biopsy sections. Antigen-retrieved sections are blocked and treated with primary, followed by secondary antibodies against S-100, a marker of Hirschsprung's disease. Chromogenic substrate is added to stain the targeted cells. Samples are dehydrated and mounted before visualizing under the microscope.

Protocol

1. Preparation of the Rectal Suction Biopsies (RSB) Sections

1. Place the rectal suction biopsies in 4% paraformaldehyde for 6 h to fix the tissue. Use a fixative volume 5 times more than the tissue volume.
2. Dehydrate the tissue using the pathological tissue dehydrator for 11 h. The whole programmed process consists of the following 5 steps:
   1. Place the tissue in formaldehyde and fix it for 1 h.
   2. Place the tissue in 75% ethanol for 4 h.
   3. Place the tissue in absolute ethanol (two changes, 1 h each).
   4. Place the tissue in absolute ethanol (two changes, 30 min each).
   5. Place the tissue in xylene (three changes, 1 h each).
3. Place the tissue in paraffin wax (58-60 °C) (three changes, 1 h each). Embed the RSB tissues in paraffin blocks.
4. Trim the paraffin blocks using a microtome until the tissue is entirely exposed with a complete section plane.
5. Cut the blocks into 0.4 μm sections with a microtome.
6. Place the sections into 45-50 °C water for a few seconds each to allow the sections to spread into a flat plate.
7. Transfer the paraffin sections onto slides.
8. Bake the slides in a 60 °C oven for 3-5 h. Avoid baking for too long, which may lead to the loss of the antigens.
9. Place the deparaffinized slides into xylene (2 changes, 15 min each).
10. Hydrate the slides in absolute ethanol, 95%, 80%, and 75% ethanol for 5 min each. Then, rinse them in reverse osmosis (RO)-purified water for another 5 min.

2. Antigen Retrieval

1. Make working dilutions for calretinin retrieval by mixing 4 mL of ethylenediaminetetraacetic acid (EDTA) antigen retrieval buffer with 200 mL of RO-purified water. Make working dilutions for S100 and Protein Gene Product 9.5 (PGP9.5) retrieval by mixing 1 mL of citric acid sodium citrate buffer (100x) with 99 mL of RO-purified water.
    NOTE: Calretinin can be retrieved preferentially by EDTA retrieval solution. However, citric acid sodium citrate buffer is more suitable for the retrieval of S100 and PGP9.5.
2. Place the slides in a coplin staining jar. Fill the jar with retrieval solution and place it into a preheated boiling water bath. After boiling for 20 min, remove the jar from the water bath and let it cool to room temperature. The cooling process takes approximately 10 min. Remove the slides and gently rinse them once with phosphate-buffered saline (PBS).
3. Draw a circle with a marker pen around the tissue and prepare a wet box for the following procedures.

3. Blocking

1. Make working dilutions by mixing 3 mL of 30% H₂O₂ with 27 mL of RO-purified water.
2. Drop two or three drops of 3% H₂O₂ solution dropwise onto the tissue to block peroxidases. Add the H₂O₂ solution promptly to ensure that it does not overflow from the circle. Block peroxidases in a 37 °C incubator for 20 min.
3. Rinse the slides gently with PBS (three washes, 5 min each).
4. Block the slides for 20 min at 37 °C with 5% bovine serum albumin (BSA, prepared by mixing 1.5 g of BSA powder with 30 mL of RO-purified water).
    NOTE: Blocking with 3% hydrogen peroxide can prevent 95% of nonspecific staining and blocking with 5% BSA can prevent the other 5% of nonspecific staining. Combine the two methods to obtain a reliable result.

4. Antigen-antibody Reaction

1. Remove the 5% BSA and add 50 μL of primary antibody (calretinin, S100, or PGP9.5) to the slide. Incubate overnight at 4 °C.
2. Wash the slide with PBS (three times, 3 min each).
3. Add 50 μL of polymer enhancer (reagent A; see Table of Materials) to the section and incubate it for 20 min in a 37 °C incubator. Then, wash the slide with PBS (three times, 3 min each).
4. Add 50 μL of secondary antibody B (goat anti-rabbit/mouse IgG secondary antibody) to the section and incubate for 30 min in a 37 °C incubator. Wash the section with PBS (three times, 5 min each).

5. 3,3-Diaminobenzidine and Hematoxylin Staining

1. Add 850 μL of RO-purified water to a 1.5 mL tube and add the staining reagents (see Table of Materials) in the order A, B, and C. Add 50 μL of each reagent to obtain a total of 1 mL of 3,3-diaminobenzidine (DAB) working solution. If there are precipitates, use the solution after filtration.
2. Add a drop of DAB working solution to the tissue section and stain for 3-10 min. Incubate the slides in DAB at room temperature until brown staining is detected. Monitor carefully using a brightfield microscope. Keep the DAB working solution away from light. Then, wash the slide with PBS for 5 min.
3. Add a drop of hematoxylin to counterstain the nuclei for approximately 1 min. Then, hold the coplin jar and wash it under a trickle of water for approximately 30-40 s until the excess dye is removed. Be careful not to damage the tissue sections.
4. Immerse the RSB slides in a coplin jar containing PBS for 25-30 s. Then, differentiate in 1% hydrochloric acid-ethanol for 10 s. Wash the slides with PBS for 1 min.
5. Dehydrate the slides in the reverse order of hydration: 75%, 80%, 95%, and absolute ethanol (5 min each).
6. Expose the slides to xylene (2 changes, 5 min each).
7. Mount the coverslip using ultraclean mounting. Allow slides to dry before visualization using a microscope.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Calretinin antibody	MXB Biotechnologies	MAB-0716 170416405c	antibody: primary antibody
S-100 antibody	MXB Biotechnologies	Kit-0007	antibody: primary antibody
PGP9.5 antibody	Shanghai long island antibody Co. Ltd	R-0457-03	antibody: primary antibody
Enhancer reagent	MXB Biotechnologies	KIT-9902-A 170416405a	antibody: secondary antibody A
Goat anti-Rabbit/Mouse IgG Secondary Antibody	MXB Biotechnologies	KIT-9902-B	antibody: secondary antibody B
DAB staining kit (containing reagent A B and C)	MXB Biotechnologies	DAB-0031	staining kit
Heat incubator	Shanghai yiheng instrument Co. Ltd	DHP-9082	instrument
Rbi2 suction rectal biopsy system	Aus Systems Pty Ltd, South Australia, Australia	CP1200 HP1000 SS1000	instrument
Microtome	Leica	leica RM2016	instrument
Citric acid sodium citrate buffer(100X)	MXB Biotechnologies	MVS-0101	antigen retrieval buffer
Pathological tissue dehydrator	Wuhan junjie electronic Co. Ltd	JT-12F	instrument