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Pharmingen

P r o t o c o l s

Preparation and Staining of Paraffin Sections

for use with Pharmingen reagents

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 3mm 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 prior to processing the tissue.
  2. 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 (New Cat. No. 550523; Previous Cat. No. 7538KZ) is helpful to preserve the antigenic epitopes.

  1. Place freshly dissected tissues trimmed 3mm thick into Zinc Fixative and allow tissues to fix for 24-48 hours at room temperature.
  2. Follow processing schedule recommended in section C.

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.

Station Time Solution
  1. - Original fixative
  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 m) on a microtome and float on a 40C 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 55C 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) (New Cat. No. 550524; Previous Cat. No. 7539KK):

  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 193F (89C) (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 193F 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 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 and 17-25 psi for 5 minutes.
  3. When completed, 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.
Note: *For methodology on other antigen retrieval systems, refer to the instructions in technical data sheets.
Note: **Heating by use of microwave oven may require a license under US patent No. 5244787.

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.

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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
ELISA/ELISPOT
Cytokine ELISA
Mouse IgE ELISA Protocol
Immunohistochemistry
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
Immunoprecipitation
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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