Preparation and Staining of Frozen Tissue Sections

For use with Pharmingen reagents

Materials neeed:

• 2-methylbutane (isopentane)
• Liquid Nitrogen
• Dry ice
• Peel-Away® base molds
• Frozen tissue matrix (OCT® or Cryomatrix®)
• Long forceps
• Necropsy tools
• Superfrost Plus slides

1. Label base mold and partially fill the mold with frozen tissue matrix.
2. Sacrifice animal by prescribed and approved euthanasia techniques.
3. Remove desired tissues, trim and cut tissue no more than 5 mm thick. Place in pre-labeled base molds filled with frozen tissue matrix. Arrange tissue in the matrix near the bottom so tissue is easily exposed when sections are cut.
4. Place a stainless steel beaker of 2-methylbutane in liquid nitrogen and allow to cool adequately. Place base mold with tissue into the beaker of cold 2-methylbutane and quickly immerse the block. Allow the tissue matrix to solidify completely and remove block from 2-methylbutane and place on dry ice or in the -20°C cryostat. NOTE: If block is left in 2-methylbutane too long, the block may crack.
5. Store blocks in the -80°C freezer until ready for sectioning.

Sectioning of Frozen Tissues

1. Before cutting sections, allow the temperature of the block to equilibrate to the temperature of the cryostat (typically -20°C).
2. Place the tissue block on the cryostat specimen disk. Adjust the positioning of the block to align the block with the knife blade. Cut tissue block until the desired tissue is exposed.
3. Cut sections of the desired thickness (usually 5 µm), place the sections on a Fisher Superfrost slide and dry overnight at room temperature (RT).
4. Fix slides by immersion in cold acetone (-20°C) for 2 minutes or other suitable fixative (e.g. alcohol, formal alcohol, formalin, etc.), air dry at RT and proceed to staining.
5. Alternatively, the frozen section slides can be stored for a short period of time at -70°C in a sealed slide box. When ready to stain, remove slides from freezer and warm to -20°C in the cryostat or -20°C freezer, fix for 2 minutes in cold fixative (acetone or other suitable fixative) and allow to come to RT to continue with the staining.

Standard Immonuhistochemical Staining Procedure for Frozen Sections

Please read entire procedure before staining sections. Perform all incubations in a humid chamber and do not allow sections to dry out. Isotype and system controls should also be run and must be matched to the isotype of each primary antibody to be tested.

Materials needed:

• Phosphate Buffered Saline (PBS) (cat. no. 554781)
• H2O2 Solution
• Antibody Diluent for IHC (Cat. No. 559148)
• Streptravidin-Horseradish Peroxidase (Cat. No. 550946)
• DAB Substrate Kit (Cat. No. 550880)
• Hematoxylin
• Bluing Reagent
• Graded alcohols
• Xylene

More conveniently, our Ig HRP detection kits can be used to perform the immunohistochemical staining. Anti-Hamster Ig (cat. no. 551012), anti-Mouse Ig (cat. no. 551011), and anti-Rat Ig (cat. no. 551013) HRP detection kits are available, and include biotinlyated secondary antibody, Antibody Diluent Buffer, Streptavidin-HRP, DAB Buffer, and DAB Chromogen.

1. Label slides with a solvent resistant pen and demarcate the tissue if required.
2. Rinse slides 3x in PBS, to remove the tissue-freezing matrix.
3. Block endogenous peroxidase activity by incubating the slides in 0.3% H2O2 solution in PBS for 10 minutes.
4. Rinse slides 3x in PBS, 2 minutes each time.
5. Block non-specific binding by incubating with blocking buffer (10% serum from host species of secondary antibody diluted in PBS or 10% FBS in PBS) for 30-60 min at RT in a humidified chamber.
6. Dilute the primary antibody in the Antibody Diluent for IHC. Alternatively, a buffered solution with a source of protein can be used as antibody diluent. Apply the diluted antibody to the tissue sections on the slide. Incubate for 1 hour at RT in a humidified chamber.
7. Rinse slides 3x in PBS, 2 minutes each time.
8. Dilute the biotinylated secondary antibody in the Antibody Diluent for IHC. Alternatively, a buffered solution with a source of protein can be used as antibody diluent. Apply to the tissue sections on the slide and incubate for 30 minutes at RT.
9. Rinse slides 3x in PBS, 2 minutes each time.
10. Apply the Streptravidin-Horseradish Peroxidase pre-diluted to the tissue sections on the slide and incubate for 30 minutes at RT.
11. Rinse slides 3x in PBS, 2 minutes each time.
12. Prepare DAB substrate solution by adding 1 drop of DAB chromagen to every 1 ml of DAB buffer. (When using other substrates follow manufacturer's recommendations.)

13. SAFETY NOTE: DAB is a suspect carcinogen. Handle with care. Wear gloves, lab coat and eye protection.

14. Drain PBS from slides and apply the DAB substrate solution. Allow slides to incubate for 5 minutes or until the desired color intensity is reached.
15. Wash 3X in water, 2 minutes each time.
16. Counterstain slides:
    
    1. Dip twice in Hematoxylin.
    2. Rinse thoroughly in water.
    3. Dip twice in Bluing Reagent or dilute ammonia water.
    4. Rinse thoroughly in water.
17. Dehydrate through 4 changes of alcohol (95%, 95%, 100% and 100%). Clear in 3 changes of xylene (or xylene substitute) and coverslip using mounting solution..