Abbkine IHC-PARAFFIN Protocol (IHC-P)

Immunohistochemical (IHC) Staining allows you to detect antigens in a tissue fixed on a glass slide. The basic steps of the IHC-P protocol are as follows:
1. Fixing and embedding the tissue
2. Cutting and mounting the section
3. Deparaffinizing and rehydrating the section
4. Antigen retrieval
5. Immunohistochemical staining
6. Counterstaining (if desired)
7. Dehydrating and stabilizing with mounting medium
8. Viewing the staining under the microscope

A: Fixation
Proper fixation is key for the success of immunohistochemistry. 10% neutral buffered formalin (NBF) is most commonly used. Other fixatives such as paraformaldehyde (PFA) or Bouin solution (formalin/picric acid) are used less frequently. The ideal fixation time will depend on the size of the tissue block and the type of tissue, but fixation between 18-24 hours seems to be ideal for most applications. Under-fixation can lead to edge staining, with strong signal on the edges of the section and no signal in the middle; over-fixation can mask the epitope. Antigen retrieval can help overcome this masking, but if the tissue has been fixed for a long period of time (i.e. over a weekend), there may be no signal even after antigen retrieval.
After fixation, the tissue block is embedded in paraffin, then cut in a microtome to the desired thickness (approximately 5 microns is ideal for IHC) and affixed onto the slide. Tissue sections are best mounted on positively charged or APES (amino-propyl-tri-ethoxy-silane) coated slides. Once mounted, the slides should be dried to remove any water that may be trapped under the section. This can be done by leaving the slide at room temperature overnight. If there is a problem with the section adhering to the slide, you may also incubate the slide at 60ºC for a few hours.

B: Deparaffinization
Before proceeding with the staining protocol, the slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section.

Materials and reagents
• Xylene
• 100% ethanol
• 95% ethanol
Method
Place the slides in a rack, and perform the following washes:
1. Xylene: 2 x 3 minutes
2. Xylene 1:1 with 100% ethanol: 3 minutes
3. 100% ethanol: 2 x 3 minutes
4. 95% ethanol: 3 minutes
5. 70 % ethanol: 3 minutes
6. 50 % ethanol: 3 minutes
7. Running cold tap water to rinse
Keep the slides in the tap water until ready to perform antigen retrieval. At no time from this point onwards should the slides be allowed to dry. Drying out will cause non-specific antibody binding and therefore high background staining.

C. Antigen retrieval
Most formalin-fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. This is due to the formation of methylene bridges during fixation, which cross-link proteins and therefore mask antigenic sites. The two methods of antigen retrieval are heat-mediated (also know as heat-induced epitope retrieval, or HIER) and enzymatic. Both antigen retrieval methods serve to break the methylene bridges and expose the antigenic sites in order to allow the antibodies to bind.
Antigen retrieval with Tris/EDTA pH 9.0 buffer is suitable for most antigens. Sodium citrate Ph 6.0 is also widely used. Heat-induced epitope retrieval is most often performed using a pressure cooker, a microwave, or a vegetable steamer. Additionally, some labs will use a water bath set to 60°C and incubate the slides in retrieval solution overnight. The optimal method for each antigen must be found experimentally.

C1. Buffer solutions for heat-induced epitope retrieval
The following solutions are three of the more popular buffers for HIER. In the absence of advice from other researchers for a particular antibody, choice of retrieval buffer is best accomplished by experiment.

• Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0)
Tri-sodium citrate (dihydrate) 2.94 g
Distilled water 1000 ml
Mix to dissolve. Adjust pH to 6.0 with 1N HCl.
Add 0.5 ml of Tween 20 and mix well. Store at room temperature for 3 months or at 4°C for longer storage.

• 1 mM EDTA, adjusted to pH 8.0
EDTA 0.37 g
Distilled water 1000 ml
Store at room temperature for 3 months.

• Tris-EDTA Buffer (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0)
Tris 1.21 g
EDTA 0.37 g
Distilled water 1000 ml (100 ml to make 10x, 50 ml to make 20x)
Mix to dissolve. pH is usually at 9.0.
Add 0.5 ml of Tween 20 and mix well. Store at room temperature for 3 months or at 4C for longer storage.

C2. Heat-induced epitope retrieval methods

Slides should be placed in a metal rack for this procedure.

Materials and reagents
• Domestic stainless steel pressure cooker
• Hot plate
• Vessel with slide rack to hold approximately 400-500 ml
• Antigen retrieval buffer (i.e. Tris/EDTA pH 9.0, sodium citrate pH 6.0)

Method
1. Add the appropriate antigen retrieval buffer to the pressure cooker. Place the pressure cooker on the hotplate and turn it on full power. Do not secure the lid of the pressure cooker at this point, simply rest it on top. While waiting for the pressure cooker to come to a boil, deparaffinize and rehydrate the sections as above.
2. Once boiling, transfer the slides from the tap water to the pressure cooker. Secure the pressure cooker lid as in the manufacturer’s instructions.
3. As soon as the cooker has reached full pressure time 3 minutes (Three minutes is only suggested as a starting point antigen retrieval time).
4. When 3 minutes has elapsed, turn off the hotplate and place the pressure cooker in an empty sink.
5. Activate the pressure release valve (see the manufacturer’s instructions) and run cold water over the cooker. Once de-pressurized, open the lid and run cold water into the cooker for 10 minutes.
6. Continue with the immunohistochemical staining protocol.
Note: Other tools like microwave also can be used according to your actual situation.

D. Immunohistochemical staining

General guidelines
All incubations should be carried out in a humidified chamber to avoid drying of the tissue. Drying at any stage will lead to non-specific binding and ultimately high background staining. A shallow, plastic box with a sealed lid and wet tissue paper in the bottom is an adequate chamber, just as long as the slides are kept off the paper and can lay flat so that the reagents don’t drain off! A good solution is to cut a plastic serological pipette into lengths to fit your incubation chamber. Glue them in pairs to the bottom of the chamber, with the 2 individual pipette tubes of each pair being placed about 4.0 cm apart. This provides a level and raised surface for the slides to rest on away from the wet tissue paper. For enzymatic methods, horseradish peroxidase (HRP) or alkaline phosphatase (AP) are the most commonly used enzymes.

Protocol
Day 1
1. (If using an HRP conjugate for detection, blocking of endogenous peroxidase can be performed here but we recommend waiting until after the primary antibody incubation. See Day 2, step 2.).
2. Wash the slides 2 x 5 minutes in TBS plus 0.025% Triton X-100 with gentle agitation.
3. Block in 10% normal serum with 1% BSA in TBS for 2 hours at room temperature.
4. Drain slides for a few seconds (do not rinse) and wipe around the sections with tissue paper.
5. Apply primary antibody diluted in TBS with 1% BSA.
6. Incubate overnight at 4°C.

Day 2
1. Rinse 2 x 5min TBS 0.025% Triton with gentle agitation.
2. If using an HRP conjugate for detection, incubate the slides in 0.3% H2O2 in TBS for 15 min.
3a. For enzymatic detection (HRP or AP secondary conjugates):
Apply enzyme-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in TBS with 1% BSA, and incubate for 1 hour at room temperature.
3b. For fluorescent detection:
Apply fluorophore-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in TBS with 1% BSA, and incubate for 1 hour at room temperature. This step should be done in the dark to avoid photobleaching.
4. Rinse 3 x 5min TBS (If using fluorescent detection, end at this step and coverslip with mounting medium; If visualizing the protein with a chromogen, continue with the following steps.).
5. Develop with chromogen for 10 min at room temperature.
6. Rinse in running tap water for 5 min.
7. Counterstain (if required).
8. Dehydrate, clear and mount.

Controls
To estimate the contribution of the non-specific interaction and Fc receptor binding, staining protocols using an antibody directed to an irrelevant antigen having the same isotype as the antibody of interest may be analyzed in parallel with the antibody of interest. The antibody directed to the irrelevant antigen is known as the isotype control. For whole serum antibodies, use normal serum from an unimmunized animal of the same species as the primary antibody. If an isotype control is not available, a negative antibody control is recommended. Simply replace the primary antibody with antibody diluent. A positive tissue control is strongly recommended to ensure that the antibody is performing as expected.
Depending on the experiment, it may also be useful to include a negative tissue control: a tissue in which the protein of interest is not expected to be found.

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