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Protocol for Immunohistochemical Staining of Paraffin Embedded Slides Using Vector Elite ABC-HRP detection

Deparaffinize and rehydrate

  • Melt paraffin from sections at 55°C for 15 minutes (optional if slides are not freshly cut).
  • Immerse slides 2x in Xylene, 2x in 100% EtOH, 1x 95% EtOH, 1x 70% EtOH, 1x 50%, 2x deionized (di) water, 5 minutes each solution.

Retrieve

  • Place slides in Abcam Antigen Retrieval Buffer (10mM citrate pH = 6.0) and heat retrieve*.
  • Rinse 1x for 1 minute in di water, 2x 3min in Tris buffered saline pH = 7.6 + 0.1% tween 20.

Quench endogenous peroxidases

  • Incubate sections in Bloxall for 10 minutes at room temperature (alternatively use freshly diluted 10% H2O2 in 1x PBS or include 10% H2O2 in MeOH during the deparaffinization steps).
  • Rinse slides in di water for 1 minute.
  • Equilibrate slides 5 minutes in buffer.

Avidin/Biotin Block

  • Wipe slides (encircle sections using a PAP pen for immunostaining if desired).
  • Place slides in humidity chamber.
  • Cover sections in Avidin reagent (Vector Labs SP-2001) and incubate 15 minutes RT.
  • Rinse avidin from slides.
  • Cover sections in Biotin reagent (Vector Labs SP-2001)) and incubate 15 minutes RT.
  • Wash 1x 5 min in Buffer at RT.

Serum block

  • Wipe slides.
  • Cover sections in 2.5% normal serum 20 minutes RT (species should match the source of the secondary antibody that will be used).
  • Do not rinse, just drain off excess block.

Antibody Incubation

  • Apply diluted primary antibody (concentration provided with website image should be initially tested along with one-fold dilution stronger and weaker).
  • Incubate 60 min at RT.
  • Wash 1x 5 min in Buffer at RT

Secondary Incubation

  • Make working ABC reagent (Vector Labs PK-6100) by combining 20µL reagent A with 20µL reagent B per 1mL of buffer and set aside. (Combined reagent must be prepared at least 30 minutes prior to use on slides.)
  • Apply Vector Labs biotinylated secondary antibody diluted 5µl/mL in buffer to sections for 30 min at RT.
  • Wash 1x 5 minutes in Buffer at RT.

ABC Incubation

  • Apply working ABC reagent to slides and incubate for 30 minutes at RT.
  • Wash 1x 5 minutes in Buffer at RT.

Detection in DAB

  • Make ImmPact DAB solution following manufacturer′s instructions. Mix well.
  • Cover sections with solution and develop while observing under a brightfield microscope.
  • Stop reaction in water rinse once desired color intensity is attained (usually 30 seconds- 2 minutes).
  • Wash 5 minutes in water.

Counterstain

  • Cover sections in OPTIK Hematoxylin for 60 seconds. Rinse well in tap water.
  • Dip slides in clarifier for 30 seconds to remove excess hematoxylin. Rinse well in tap water.
  • Dip slides in bluing solution for one minute and rinse in tap water to intensify blue color of counterstain.
  • Run slides back through graded ethanols to xylenes. Three xylene stations are recommended.

Mount sections with DPX or other permanent mountant.

Notes:
-Do not allow slides to become dry at any point during this assay.
-Tissues tested are primarily 4% PFA perfused and 24 hour immersion fixed prior to paraffin processing and embedding.
-When wiping slides, tip buffer away from sections and avoid contact with the tissue.

*We standardly use a pressure cooker for 15 min followed by a 10 minute bench cool down, but any lab standardized citra retrieval method would be appropriate.

©EnCor Biotechnology Inc.