Prestige Antibody® Immunohistochemistry Procedure

IHC Protocol

Prestige Antibodies® developed for immunohistochemistry based expression profiling are recommended to be used 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®

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 xylene**
  17. Coversliping.

* For polyclonal antibodies disregard steps 6 and 7.

** Steps 14 -16 are performed in a histostaining instrument (Leica Autostainer XL).

Reagents

  • Wash buffer (10x concentrate). Working solution originally contains 0.05% (v/v) Tween 20. Extra Tween 20 is added to a final concentration of 0.20%.
  • Retrieval Solution: Citrate buffer®, pH 6.
  • Antibody diluent.
  • Mayer’s hematoxylin
  • Xylene

Alternative Antigen Retrieval Method

For selected antibodies, alternative retrieval buffers and/or enzymatic antigen retrieval may have been used as stated on the Product Datasheet.

Enzymatic Antigen Retrieval

Enzymatic retrieval is performed in the immunostaining instrument and refers to incubation of TMA-slides in Proteinase K for 10 minutes at room temperature.

Heat Induced Epitope Retrieval (Hier) In Retrieval Buffer pH 9

HIER in retrieval buffer pH 9 is performed as the standard HIER except that retrieval buffer with pH 9 is used instead of retrieval buffer with pH 6.

Revised October 2017

 

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