Immunohistochemistry Protocols IHC Protocols

Sample preparation

Frozen tissue preparation 1: snap freezing followed by fixation

Note that the optimal fixation conditions will depend on the specific tissue and organism, which must be determined empirically.

1. Dissect the tissue of interest (<10 mm in size) and place in a pre-labeled tissue mold.
2. Cover the tissue sample in cryo-embedding media (OCT).
3. Freeze the tissue block by slowly submerging in liquid nitrogen or by placing on top of dry ice. Allow the sample to freeze completely.
4. Store the frozen tissue block at -80°C until ready for sectioning.
5. Prior to sectioning, transfer the tissue block to the cryostat set at -20°C and allow the block to equilibrate for 15 minutes.
6. Section the tissue block into ~6 – 15 μm thick sections and transfer the sections onto positively charged glass slides.
7. Dry the sections at room temperature briefly, then fix using an appropriate fixation method. For example, fix sections in ice-cold acetone for 10 minutes.
8. Wash the slides in PBS two times for 5 minutes each.
9. The slides can be stored at -80°C for several months.
10. Proceed with Fluorescence-conjugated antibody staining or Enzyme-conjugated chromogenic antibody staining.

Frozen tissue preparation 2: fixation followed by freezing

1. Fix the tissue by perfusing the organism with 4% paraformaldyhde or dissect the tissue and immerse it in 4% paraformaldehyde for 2 – 24 hours at 4°C or room temperature.
2. Perform a cryoprotection step by perfusing the organism or immersing the dissected tissue overnight at 4°C in a solution of 30% sucrose in PBS. Longer incubations may be needed for larger tissues; the tissue will sink once equilibrated with the sucrose solution.
3. Once cryopreserved, the tissue can be sectioned immediately or embedded in OCT and stored at -80°C until ready for sectioning.
4. Section the tissue block into ~6 – 30 μm thick sections and transfer the sections onto positively charged glass slides, which can be stored at -80°C for several months. Thicker sections can be transferred to wells containing PBS for free-floating IHC.
5. Proceed with Fluorescence-conjugated antibody staining or Enzyme-conjugated chromogenic antibody staining.

Formalin-fixed, paraffin-embedded (FFPE) tissue sections

1. Following perfusion or immersion fixation of the tissue, rinse the tissue in PBS.
2. Dehydrate the tissue in a series of ethanol solutions for 30 minutes each:
1. 50% ethanol
2. 70% ethanol
3. 80% ethanol
4. 95% ethanol
5. 100% ethanol
6. 100% ethanol
7. 100% ethanol
3. Clear the tissue in three changes of xylene for 20 minutes each.
4. Infiltrate the tissue with paraffin by incubating with three changes of 60°C paraffin for 1 hour each (can be overnight, though this may increase the risk of tissue cracking during sectioning).
5. Embed in a paraffin block, orienting the tissue as desired. Once solidified, the block may be stored at room temperature for several years.
6. Section the paraffin-embedded tissue into 5 – 15 μm slices on a microtome and transfer sections to silanized or gel-coated slides.
7. Dry the slides overnight at room temperature. The slides can be stored at room temperature.
8. Once ready for immunostaining, deparaffinize the slides in two changes of xylene for 3 minutes each.
9. Rehydrate the slides in a series of ethanol solutions for 3 minutes each:
1. 100% ethanol
2. 100% ethanol
3. 95% ethanol
4. 70% ethanol
5. 50% ethanol
10. Rinse the slides in running water for 10 minutes to remove ethanol. Do not allow the slides to dry out for the rest of the protocol.
11. If required, perform Antigen retrieval.
12. Proceed with Fluorescence-conjugated antibody staining or Enzyme-conjugated chromogenic antibody staining.

Antigen retrieval

Heat-induced epitope retrieval (HIER)

HIER can be performed using one of the retrieval buffers below. In all cases, the retrieval time may be shortened or extended as necessary in 5-minute increments, between 5 – 30 minutes. All boiling steps can be performed in a pressure cooker, microwave, or steamer. Alternatively, the slides can be incubated in the retrieval buffer in a 60°C water bath overnight.

1. Citrate buffer
1. Boil slides in 10 mM sodium citrate buffer (pH 6.0) and maintain at approximately 98°C for 20 minutes.
2. Cool the slides completely before moving on to the next step. Proceed with immunostaining.
2. EDTA buffer
1. Boil slides in 1 mM EDTA (pH 8.0) and maintain at approximately 98°C for 15 minutes.
2. Cool the slides completely before moving on to the next step. Proceed with immunostaining.
3. Tris-EDTA buffer
1. Boil slides in 10 mM Tris/1 mM EDTA (pH 9.0) and maintain at approximately 98°C for 20 minutes.
2. Cool the slides completely before moving on to the next step. Proceed with immunostaining.

Protease-induced epitope retrieval (PIER)

Ensure all sections are treated with enzyme for the same length of time. The digestion time may be shortened or extended as necessary.

1. Using a hydrophobic pen, draw a circle around each tissue section.
2. Prepare the protease working solution (for example, 0.05% trypsin in 0.1% calcium chloride (pH 7.8) or 0.5% pepsin in 10mM HCl (pH 2.0)).
3. Apply the protease solution to the tissue sections and incubate in a humidity chamber at 37°C for 10 minutes.
4. Rinse the slides in running water for 3 minutes.
5. Proceed with immunostaining.

Staining and detection

Most steps can be applied to sections already on slides, free-floating sections, and wholemount tissue. Steps that are specific to one type of preparation are indicated.

Fluorescence-conjugated antibody staining

Note that the optimal fixation conditions will depend on the specific tissue and organism, which must be determined empirically.

Blocking non-specific binding

1. For sections on slides draw a border around each tissue section or around all sections collectively using a hydrophobic pen
2. Permeabilize the tissue by adding a sufficient volume of wash buffer (1X TBS or PBS plus 0.025% Triton X-100) to the tissue sections within the hydrophobic barriers and incubate for 10 minutes at room temperature.
3. Block non-specific binding by incubating with blocking buffer for 1 hour at room temperature in the humidity chamber.

Antibody staining

1. Dilute the primary antibody in blocking buffer and incubate the tissue sections with the antibody solution overnight at 4°C in the humidity chamber. The optimal antibody dilution must be determined empirically, typically by dilution series.
2. Wash sections three times with wash buffer for 10 minutes each.
3. Dilute the fluorescently conjugated secondary antibody in blocking buffer and incubate the tissue sections with the secondary antibody solution for 1 – 2 hours at room temperature. Consult the antibody datasheet for a recommended dilution range, but it is typically 1:500 – 1:1000 from commercial sources.
4. Wash sections three times with wash buffer for 10 minutes each.

Counterstaining and mounting

1. If desired, incubate the tissue sections with a counterstain according to the manufacturer’s instructions. For example, to stain double-stranded DNA in cell nuclei, DAPI can be diluted to 0.5 μg/mL and incubated with the tissue sections for 5 minutes at room temperature, followed by a 5-minute wash.
2. Free-floating sections can now be carefully moved onto slides, typically with a fine paintbrush, and left to partially air dry so that they remain adhered to the slide. If wholemount tissue is flat and thin enough (~<100 μm thick), such as mouse brain slices or intestinal muscle, it may be mounted on slides. If not, the tissue will need to be held and stabilized by different means, such as embedding in agarose or using glass-bottomed imaging wells, but this will be highly tissue- and application-specific.
3. Rinse slides in ddH₂O to remove excess salts from wash buffer.
4. Dab away excess moisture from the slides using a tissue.
5. Apply anti-fade mounting medium to preserve fluorescent signal, and mount coverslips over the tissue sections. Seal the coverslips using nail polish to stabilize the cover slip (particularly if using an inverted microscope) and prevent drying out long term.
6. Proceed with microscopy analysis of the tissue.
7. Store the slides at 4°C in a slide box.

Enzyme-conjugated chromogenic antibody staining

Note that the optimal fixation conditions will depend on the specific tissue and organism, which must be determined empirically.

Blocking non-specific binding

1. Perform the blocking step as in the fluorescently conjugated antibody staining protocol. If using an HRP conjugated antibody, use TBS instead of PBS in all immunostaining reagents.

Antibody staining and chromogenic detection

1. Dilute the primary antibody as appropriate in blocking buffer and incubate overnight at 4°C in the humidity chamber.
2. Wash twice with TBS wash buffer for 10 minutes each.
3. If using an HRP conjugated antibody for detection, incubate the samples with 0.3% hydrogen peroxide in TBS for 15 minutes at room temperature.
4. Dilute the biotinylated secondary antibody in TBS blocking buffer and incubate for 1 hour at room temperature.
5. Rinse the slides three times for 5 minutes each in TBS wash buffer.
6. If using the avidin-biotin complex (ABC) method, prepare the reagent according to the manufacturer’s instructions.
7. Incubate the sections with ABC reagent for 30 minutes at room temperature.
8. Wash the slides three times for 5 minutes each in TBS wash buffer.
9. Incubate the sections with the chromogenic HRP substrate 3,3’-diaminobenzidine (DAB) for roughly 10 minutes at room temperature, or until color develops. Check regularly to prevent over-developing.
10. Rinse the slides in three changes of distilled water for 3 minutes each.

Counterstaining and mounting

1. Counterstain the slides with hematoxylin (blue, nuclei), nuclear fast red, or other counterstains if necessary.
2. If chromogenic detection is being performed with DAB, NBT, or other organic chromogens, dehydrate the sections for 10 seconds each:
1. 95% ethanol
2. 95% ethanol
3. 100% ethanol
4. 100% ethanol
5. Xylene
6. Xylene
3. Mount the sections with coverslips using an aqueous or organic mounting medium suitable for the chromogenic stains.

Buffers and Reagents

General

1X PBS

• 6.46 mM Na₂HPO₄.2H₂O
• 1.49 mM NaH₂PO₄.2H₂O
• 137 mM NaCl
• 2.68 mM KCl

0.1 M Phosphate buffer

• 108 mM Na₂HPO₄.7H₂O
• 25.3 mM NaH₂PO₄.H₂O

Fixatives

4% Paraformaldehyde (PFA)

• 4 g PFA powder
• 10 μl 10 N NaOH
• 100 ml 0.1 M phosphate buffer

Heat phosphate buffer in a glass container in microwave for 30 – 60s (do not boil). Transfer to a heated stir plate (~65-75°C) in a fume hood and add stir bar and NaOH. Add 4 g of PFA to the heated solution and leave stirring for several minutes until all of the PFA has dissolved (there may be a few granules left undissolved). Cool and filter, then store at 4°C.

Methanol

• 100% Methanol (-20°C)

Methanol/Acetone

• 50% Methanol (-20°C)
• 50% Acetone (-20°C)

Antigen retrieval buffers

Sodium citrate buffer (pH 6.0)

• 10 mM sodium citrate
• 0.05% Tween 20

EDTA buffer (pH 8.0)

• 1 mM EDTA

Tris-EDTA buffer (pH 9.0)

• 10 mM Tris base
• 1 mM EDTA
• 0.05% Tween-20

Trypsin working solution (pH 7.8)

• 0.05% Trypsin
• 0.1% CaCl₂

Pepsin working solution (pH 2.0)

• 0.5% pepsin
• 10 mM HCl

Blocking solutions

Serum block

• 1X PBS
• 1-5% normal donkey or goat serum (depending on host of secondary antibodies)
• 0.1-0.3% Triton X-100

BSA block

• 1X PBS
• 1% BSA
• 0.1-0.3% Triton X-100

Antibody diluent

• 1X PBS
• 1% BSA
• 0.1-0.3% Triton X-100

Wash buffers

Triton X-100

• 1X PBS
• 0.1% Triton X-100

Tween-20

• 1X PBS
• 0.05% Tween-20