Vectastain universal kit hrp

Tumor tissue samples were collected from xenograft tumors, fixed in 10% neutral buffered formalin, paraffin-embedded, and cut into sections. The sections were heated in 10 mM citrate buffer (pH 6.0) for EGFR, HER2, and Ki67 or in EDTA buffer (pH 8.0) for HER3 with a Decloaking Chamber. Samples were incubated with rabbit anti-EGFR (Abcam; ab52894, 1:200), anti-HER2 (Cell Signaling; cs4290, 1:200), anti-HER3 (Cell Signaling; cs12708, 1:50), or anti-Ki67 (Cell Signaling; cs9027, 1:800). Sections were stained by VECTASTAIN universal kit/HRP (Vector Lab, Burlingame, CA). Antibody binding was revealed by addition of 3,3′-diaminobenzidine substrate. Tissues were counterstained with Mayer’s hematoxylin (Thermo Fisher Scientific, Waltham, MA) and were examined using an Olympus BX51 microscope.

Tumors were processed for immunohistochemistry as previously described (24 (link)). Ki67 (CST #9027, 1:400) antibody was used and bound antibodies were detected using the VECTASTAIN Universal Kit/HRP (Vector Laboratories) and 3,3’-diaminobenzidine substrates. Images are shown at a magnification of 20X and were quantified in three separate areas by counting the number of positive cells and creating an average and standard error that is presented in graphic form.

Tumors were processed for immunohistochemistry (IHC) as previously described (50 (link)). Sections were heated in 10mmol/L citrate buffer (pH 6.0) or 10mmol/L Tris+1mmol/L EDTA buffer (pH 9.0) with a decloaking chamber and incubated overnight at 4ºC with pS6rpY235/236 (1:200), Ki67 (1:800), or MERTK (1:50) antibodies. Bound antibodies were detected using the VECTASTAIN Universal Kit/HRP (Vector Laboratories) and 3,3’-diaminobenzidine substrates. Tissues were counterstained with Mayer hematoxylin (ThermoFisher Scientific) and were examined using an Olympus BX51 microscope. Images are shown at a magnification of 20X.