Vectastain abc reagent

IHC staining of breast cancer tissue was performed for ERRβ. Five micron sections from formalin fixed paraffin embedded tissues were de-paraffinized with xylenes and rehydrated through a graded alcohol series. Heat induced epitope retrieval (HIER) was performed by immersing the tissue sections in Target Retrieval Solution, Low pH (DAKO) in the PT Link (DAKO). IHC staining was performed using the VectaStain Kit from Vector Labs according to manufacturer’s instructions. Briefly, slides were treated with 3% hydrogen peroxide, avidin/biotin blocking, and 10% normal goat serum and independently exposed to primary antibodies for ERRβ2- cl .07, 1:150, 1:240 (R&D systems, #PP-H6707-00) and ERRβsf- cl .05, 1:150 (R&D systems, #PP-H6705-00) for 1 hour at room temperature. Slides were exposed to appropriate biotin-conjugated secondary antibodies (Vector Labs), Vectastain ABC reagent and DAB chromagen (Dako). Slides were counterstained with Hematoxylin (Fisher, Harris Modified Hematoxylin), blued in 1% ammonium hydroxide, dehydrated, and mounted with Acrymount. Control tissues with the primary antibody omitted were used as negative controls. Images of the full TMA slide stained for of Hematoxylin and eosin, ERRβsf- cl .05, and ERRβ2- cl .07 are available on figshare (https://doi.org/10.6084/m9.figshare.9992891.v1).

Immunohistochemistry was performed in the Georgetown University Medical Center Histopathology and Tissue Shared Resource. Five micron sections from formalin fixed paraffin embedded tissues were de-paraffinized with xylene and rehydrated through a graded alcohol series. Heat induced epitope retrieval (HIER) was performed by immersing the tissue sections at 98°C for 20 minutes in Target retrieval solution, high pH (Dako). Immunohistochemical staining was performed using the VectaStain Kit from Vector Labs according to manufacturer’s instructions. Briefly, slides were treated with 3% hydrogen peroxide for 10 minutes. Endogenous biotin was blocked using an avidin/biotin blocking kit from Invitrogen. The slides were then treated with 10% normal goat serum for 10 minutes and exposed to primary antibody for STAG2 (1:50, Santa Cruz, sc81852) for 1 hour at room temperature. Slides were then exposed to biotin-conjugated mouse secondary antibody (Vector Labs), Vectastain ABC reagent and DAB chromagen (Dako). Slides were counterstained with hematoxylin (Fisher, Harris Modified Hematoxylin) at a 1:8 dilution for 2 minutes at room temperature, blued in 1% ammonium hydroxide for 1 minute at room temperature, dehydrated, and mounted with Acrymount.