PAP/APAAP Immunostaining of Paraffin- Embedded Tissue Sections

PAP/APAAP immunostaining of paraffin embedded tissue sections

PAP/APAAP immunostaining of paraffin embedded tissue sections

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For use with unconjugated monoclonal and polyclonal antibodies.

This method provides a general procedure for use with the majority of Bio-Rad reagents. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with product and batch specific information provided with each vial. Please note that a certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques - these are guidelines only and may need to be adjusted for particular applications.


1. 0.3% H2O2‎, 70% methanol in TBS
1 ml of 30% H2O2‎ per 100 ml methanol/TBS.
2. TBS stock solution (10x)
NaCl, 87.66 g
Tris base, 60.55 g
Distilled water, 1 liter
Adjust pH to 7.4 using concentrated HCl


  1. De-paraffinize sections thoroughly in xylene/synthetic solvent, and hydrate through a graded series of alcohols. Wash twice in water.
  2. Outline section with PAP pen.
  3. If required, treat with 0.3% (w/v) H2O2‎ in methanol for 15 minutes to block endogenous peroxidase activity (2% (w/v) H2O2‎/methanol can be used for a shorter time if preferred). Bio-Rad offers peroxide blocking reagent (BUF017B). Wash 3 times in TBS.
  4. If required, include an appropriate antigen retrieval step to enhance the immunostaining (see Protocol: Antigen Retrieval Techniques for use with Formalin-Fixed Paraffin-Embedded Sections). Wash once in TBS.To make antigen retrieval as easy as possible, Bio-Rad offers a variety of retrieval/antigen unmasking fluids. Wash once in water.
  5. Incubate sections for 10 minutes in 10% normal serum from the species in which the secondary antibody was raised. Tap excess serum off the slides before staining.
  6. Incubate sections with primary antibody for at least 1 hour at room temperature in a humid chamber or overnight at 4°C. Wash three times in TBS.
  7. Add bridging secondary antibody at recommended dilution (see specific datasheet for details). Incubate for at least 30 minutes at room temperature. Wash three times in TBS.
  8. Add enzyme complex at recommended dilution (see specific datasheet for details). Incubate for at least 30 minutes at room temperature. Wash three times in TBS.
  9. Incubate with the appropriate substrate solution for the recommended period of time (Bio-Rad suggests the use of DAB substrate with HRP-conjugated streptavidin, and Fast Red/Napthol AS-MX for Alkaline Phosphatase-conjugated streptavidin). Wash once in water.
  10. Counterstain with hematoxylin for 1 minute. “Blue” with running water for 5 minutes. Then wash.
  11. Mount in aqueous mounting medium, or alternatively dehydrate through a graded series of alcohols and xylene/solvent. Mount in synthetic mountant.


  • Do not allow slides to dry out after the fixation step, as drying will result in damage to the tissue structure.
  • Beware, certain substrates are soluble in alcohol – please refer to supplier information for details.
  • Appropriate controls should always be carried out. It may be useful to include a sample in which no primary antibody is used at all, in order to determine any non-specific binding of the secondary reagent to the target tissue.

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