Prestige Antibodies® developed for immunohistochemistry based expression profiling are recommended to be used according to the standard IHC staining protocol described below.
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.
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.
All incubations are performed at room temperature.
All reagents are applied at a volume of 300 µL per slide.
* For polyclonal antibodies disregard steps 6 and 7.
** Steps 14 -16 are performed in a histostaining instrument (Leica Autostainer XL).
For selected antibodies, alternative retrieval buffers and/or enzymatic antigen retrieval may have been used as stated on the Product Datasheet.
Enzymatic retrieval is performed in the immunostaining instrument and refers to incubation of TMA-slides in Proteinase K for 10 minutes at room temperature.
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