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P r o t o c o l s

Preparation and Staining of Frozen Tissue Sections

for use with Pharmingen reagents

I. Preparation of Frozen Sections for Sectioning

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 -20C cryostat. NOTE: If block is left in 2-methylbutane too long, the block may crack.
  5. Store blocks in the -80C freezer until ready for sectioning.

II. Sectioning of Frozen Tissues

  1. Before cutting sections, allow the temperature of the block to equilibrate to the temperature of the cryostat (-20C).
  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 m), place the sections on a Fisher Superfrost slide and dry overnight at room tem-perature.
  4. Fix slides by immersion in cold acetone (-20C) for 2 minutes or other suitable fixative (e.g. alcohol, formal alcohol, formalin, etc.), air dry at room temperature and proceed to staining (Section III).
  5. Alternatively, the frozen section slides can be stored for a short period of time at -70C in a sealed slide box. When ready to stain, remove slides from freezer and warm to -20C in the cryostat or -20 freezer, fix for 2 minutes in cold fixative (acetone or other suitable fixative) and allow to come to room temperature to continue with the staining.

III. 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)
  • H2O2 Solution
  • Antibody Diluent for IHC (Cat. No. 559148/70991A)
  • Streptravidin-Horseradish Peroxidase (Cat. No. 550946/75477E)
  • DAB Substrate Kit (Cat. No. 550880/7578KK)
  • Hematoxylin
  • Bluing Reagent
  • Graded alcohols
  • Xylene

More conveniently, our Ig HRP detection kits can be used to perform the immunohistochemical staining.

  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. 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 room temperature in a humidified chamber.
  6. Rinse slides 3x in PBS, 2 minutes each time.
  7. 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 room temperature.
  8. Rinse slides 3x in PBS, 2 minutes each time.
  9. Apply the Streptravidin-Horseradish Peroxidase pre-diluted to the tissue sections on the slide and incubate for 30 minutes at room temperature.
  10. Rinse slides 3x in PBS, 2 minutes each time.
  11. Prepare DAB substrate solution by adding 1 drop of DAB chromagen to every 1 ml of DAB buffer. (When using other substrates follow manufacturers recommendations.)

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

  12. 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.
  13. Wash 3X in water, 2 minutes each time.
  14. 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.
  15. Dehydrate through 4 changes of alcohol (95%, 95%, 100% and 100%). Clear in 3 changes of xylene (or xylene substitute) and coverslip.
Protocols Index>>
Flow Cytometry
Fluorochrome Absorption and Emission Spectra
Procedure for Setting Compensation for Multi-Color Flow Cytometric Analysis
Cell Fixation/Permeabilization Kits
Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis
Annexin V Staining Protocol
APO-BRDU™ Procedure
APO-DIRECT™ Procedure
Detection of BrdU Incorporation in DNA Synthesizing Cells
Staining Procedure for Flow Cytometric Detection of Human Cyclins
Indirect Immunofluorescence Staining of Human Platelets
Immunofluorescence Staining of Human Cells by Lysed Whole Blood Method
Immunofluorescent Staining of Mouse and Rat Leukocytes
The Uses of Fc Block in Immunophenotyping of Mouse or Rat Leukocytes
Cytokine ELISA
Mouse IgE ELISA Protocol
Preparation and Staining of Frozen Tissue Sections
Preparation and Staining of Paraffin Sections
Microwave citrate Pretreatment of Paraffin Sections
Immunohistochemistry / Tissue Section Staining
Cell Staining for Immunofluorescence Microscopy
Western Blotting
Western Blotting with Alkaline Phosphatase Conjugates
Western Blotting with Biotinylated Antibodies
Western Blotting with Horseradish Peroxidase Conjugates
Western Blotting with Monoclonal Antibodies
Western Blotting with Rabbit Polyclonal Antibodies
Preparation of Brain Membrane Fractions for Western Blot Analysis
Immunopurification of Tyrosine Phosphorylated Proteins
Immunoprecipitation with Antibody:Agarose Conjugates
Immunoprecipitation with anti-Phosphotyrosine: Biotin Conjugates
Immunoprecipitation With Soluble Antibodies
Immunoprecipitation of 35S Labeled Cells
RNAse Protection Assay (RPA)
RNase Protection Assay
Total RNA Isolation
Protein Kinase Assay (PKA)
Protein Kinase Assay with an Immunocomplex
Protein Kinase Assay after Denaturation and Renaturation

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