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P r o t o c o l s

Microwave citrate Pretreatment of Paraffin Sections





Deparaffinize slides a (after drying thoroughly overnight at RT) in 2 changes of xylene (or xylene substitute) for 10 mins each.








Transfer slides to 100% alcohol, 2 changes for at least 2 min each.








Block endogenous peroxidase activity by incubating for 10 min in 3% H2O2.








Rinse 2-3 times in water.








Place slides in a plastic coplin jar or Tek® staining dish and fill container with 10 mM citrate bufferb, c , pH 6.0.








Place staining dish in microwave with inverted lid on top. If using a probe, set temperature to 193°F. (If your microwave does not have a probe, see reference below.)








Mix the solution with a disposable pipet after temperature has been reached and hold that temperature in the microwave for 10 min.








Remove slides from microwave and set the covered dish on the counter for an additional 20 min.








Rinse slides 2-3 times in PBS and continue with Step 10 of "Preparation of Paraffin Sections"


a. For best adherence, use Superfrost Plus® slides (Poly-L-lysine or silane slides are also acceptable) and use only deionized water in the waterbath when cutting paraffin sections.

b. When staining paraffin-embedded sections, antigens are affected differently by the various methods of pretreatment (citrate buffer, trypsin, or no pretreatment). Please call Technical Service for specific information about antigen retrieval. It should be noted that certain antigenic specificities cannot be detected in paraffin-embedded sections.

c. Preparation of 10 mM Citrate Buffer (pH 6.0)

Stock A Citrate Buffer

Stock B Citrate Buffer

Working 10 mM Citrate Buffer

4.2 g Citric Acid

14.7 g Sodium Citrate

18 ml Stock A

20 mls ddH20

500 mls ddH20

82 ml Stock B



Fill to 1 liter with ddH20 and pH to 6.0

1. Tacha, DE, Chen T. Modified antigen retrieval procedure: calibration techniques for microwave ovens. Histotechnology 17(4):365,1994.

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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 Blockô 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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