Prestige Antibody® Immunohistochemistry Procedure

IHC Protocol

Prestige Polyclonals and Monoclonals developed for immunohistochemistry based expression profiling are recommended to use according to the standard IHC staining protocol described below.

Deparaffinization

Paraffin sections of 4 μm thickness are baked  overnight at 50°C. Prior to immunostaining, deparaffinization and hydration is performed in xylene and graded ethanol to distilled water. During hydration, a 5 minutes blocking for endogenous peroxidase is performed in 0.3% H2O2 in 95% ethanol.

Standard Antigen Retrieval Method

The standard antigen retrieval method is Heat Induced Epitope Retrieval (HIER) in retrieval buffer pH 6, using a pressure boiler (Decloaking chamber, Biocare Medical, Walnut Creek, CA, USA) as heat source. HIER is performed by heating the TMA-slides immersed in retrieval buffer for 4 minutes at 125°C in the pressure boiler. After completed boiling, slides remain in the pressure boiler and are allowed to cool to 90°C. The total processing time is approximately 45 minutes.

NOTE: The specified working dilutions of the primary antibodies are to be considered as a guideline only. Optimal dilutions must be determined by the user.

Immunohistochemical Staining Program, Autostainer 480®

(ThermoFisher Scientific, Waltham, MA, USA)

All incubations are performed at room temperature.
All reagents are applied at a volume of 300 μl per slide.

  1. Rinse in wash buffer.
  2. Incubation with Ultra V Block for 5 min.
  3. Rinse in wash buffer (x2).
  4. Incubation with primary antibody for 30 min.
  5. Rinse in wash buffer (x3).
  6. Incubation with Primary Antibody Enhancer for 20 min.*
  7. Rinse in wash buffer (x2).*
  8. Incubation with labeled polymer for 30 min.
  9. Rinse in wash buffer (x2).
  10. Developing in DAB solution for 5 min.
  11. Rinse in distilled water.
  12. Counterstaining in hematoxylin for 5 min.**
  13. Rinse in tap water for 5 min.**
  14. Rinse in lithium carbonate water, diluted 1:5 from saturated solution for 1 min.**
  15. Rinse in tap water for 5 min.**
  16. Dehydration in graded ethanol and xylen**
  17. Coversliping.

* For polyclonal antibodies disregard steps 6 and 7.
** Steps 14 -16 are performed in a histostaining instrument (Leica Autostainer XL).

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