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