Protocols

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Preparation and Staining of Paraffin Sections

I. Fixation and Processing of Tissue for Paraffin Sections

A. Fixation of Tissues in 10% Neutral Buffered Formalin

1. Sacrifice animal by prescribed and approved euthanasia techniques. Tissues to be fixed and processed should be cut to a size no larger than 3 mm thick. Let tissues fix in 10% formalin at room temperature for 8 hours but not to exceed 24 hours. (For small rodent tissue, it is recommended to fix tissues for 4-8 hours.)
2. Rinse with running tap water for 1 hour.
3. Follow processing schedule recommended in section C.

B. Fixation of Tissues in Zinc Fixative:

Many antigenic epitopes are masked or even destroyed by 10% formalin fixation . In some cases fixation in a milder fixative such as Zinc fixative for IHC (cat. no. 550523) is helpful to preserve the antigenic epitopes.

1. Place freshly dissected tissues trimmed 3 mm thick into Zinc Fixative and allow tissues to fix for 24-48 hours at room temperature.
2. Rinse with running tap water for 30-45 minutes.
3. Follow processing schedule recommended in section C, step 2.

C. Processing Schedule:

Note: The processing, embedding and sectioning of paraffin blocks requires specialized equipment and expertise and is usually performed by a histology or pathology laboratory. While hand processing can be performed according to the following protocol the results may show marked variation in histology quality and antigenicity.

1. Water
2. 45 minutes 70% Alcohol
3. 45 minutes 80% Alcohol
4. 45 minutes 95% Alcohol
5. 45 minutes 100% Alcohol
6. 60 minutes 100% Alcohol
7. 60 minutes 100% Alcohol
8. 60 minutes Clearing Reagent (xylene or substitute)
9. 60 minutes Clearing Reagent (xylene or substitute)
10. 60 minutes Paraffin 1
11. 60 minutes Paraffin 2
12. 60 minutes Paraffin 3

II. Preparation of Paraffin Sections for Immunohistochemistry

A. Sectioning Protocol:

1. Section paraffin blocks at the desired thickness (usually 4-5 µm) on a microtome and float on a 40°C water bath containing distilled water.
2. Transfer the sections onto a Superfrost Plus slide. Allow the slides to dry overnight and store slides at room temperature until ready for use.

B. Deparaffinization and re-hydration of tissue slide:

1. Before deparaffinization, place the slides in a 55°C oven for ten minutes to melt the paraffin. Deparaffinize slides in 2 changes of xylene or xylene substitute for 5 minutes each.
2. Transfer slides to 100% alcohol, 2 changes for 3 minutes each and transfer once through 95% alcohol for 3 minutes.
3. Block endogenous peroxidase activity by incubating sections in 3% H₂O₂ solution in methanol for 10 minutes.
4. Rinse in PBS 2X for 5 minutes each time.
5. If the antibody staining requires antigen retrieval to unmask the antigenic epitope refer below to section C. If antigen retrieval is not required proceed to section D.

C. Pretreatment of paraffin sections with BD Retrievagen A* (pH 6.0) (cat. no. 550524):

1. Make a working solution of Retrievagen A by mixing 18 ml of Retrievagen A solution 1 and 82 ml of Retrievagen A solution 2 and bring the final volume to 1 liter in distilled water.
2. Place slides in a plastic coplin jar filled with the working Retrievagen solution and heat in a microwave oven to 203°F (95°C) (microwave oven ** or other heating sources such as pressure cooker (see alternate protocol), water bath can be used).
3. Mix the working Retrievagen A solution in the coplin jar with a disposable pipet and incubate the slides at 203°F for 10 minutes.
4. Remove the coplin jar with the slides, cover the jar tightly, and allow the solution to slowly cool to room temperature for 20 minutes.
   Note: It is important to let the temperature ramp down slowly to allow the protein molecules to fold properly.
5. Rinse slides in PBS 3X, 5 minutes each time.

Alternate Protocol

1. For antigen retrieval using a pressure cooker or an autoclave, pretreat slides with BD Retrievagen A solution in a glass or metal coplin jar as outlined in step C1 above.
2. Heat coplin jar containing slides with BD Retrievagen A solution in a pressure cooker or autoclave at 120-125°C, 17-25 psi for 5 minutes.
3. When completed (at 0 psi), open pressure cooker or autoclave and allow slides to cool to room temperature (approximately 20-30 minutes) prior to removing them from the coplin jar.
4. Wash slides as indicated in step C5 above.

D. Immunohistochemical staining of paraffin embedded tissues:

Refer to "Standard Immunohistochemical Staining Procedure" (Section III of Immunohistochemical staining of frozen sections).
Begin at step 5 and proceed through coverslipping.