Vectastain universal secondary antibody

Paraffin-embedded tissue sections were deparaffinized, rehydrated, subjected to citrate buffer antigen retrieval, permeabilized with 2N HCl, and then blocked with 5% horse serum. Sections were then stained with a monoclonal rabbit antibody against pAKT serine 473 (1:50 in 5% horse serum in PBS, antibody #3787; Cell Signaling Technology, Danvers, Massachusetts, USA) or a polyclonal rabbit antibody against pS6 ribosomal protein Ser235/236 (1:50 in 5% horse serum in PBS, antibody #2211; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. Each slide was then washed and incubated with VectaStain universal secondary antibody (1:50 in 5% horse serum in PBS; Vector Laboratories, Inc., Burlingame, California, USA). Slides were then subjected to R.T.U. VectaStain ABC reagent (Vector Laboratories, Inc., Burlingame, California, USA). Finally, sections were incubated with 3,3′-diaminobenzidine substrate (DAB Peroxidase Substrate; Vector Laboratories, Inc., Burlingame, California, USA); counterstained with hematoxylin (Hematoxylin QS, Vector Laboratories, Inc., Burlingame, California, USA); dehydrated; and mounted. Light microscopy was performed and images at ×200 magnification were acquired using the Zeiss Axio Imager M2 imaging system. Images were then analyzed via ImageJ with measurements taken in RawIntDen/Area (raw integrated density/area) of the intensity signal.

The protein expression of phosphorylated-Serine473-AKT (pAKT) and phosphorylated S6 (pS6) were assessed using immunohistochemistry, as they are downstream products of PI3K and mTOR activation, respectively. Paraffin-embedded sections were deparaffinized, rehydrated, subjected to citrate buffer antigen retrieval, permeabilized with 2N HCl, and then blocked with 5% horse serum. Sections were then stained with a monoclonal rabbit antibody against pAKT (1:50 in 5% horse serum in phosphate buffered saline (PBS); antibody #3787, Cell Signaling Technology) or a polyclonal rabbit antibody against pS6 (1:50 in 5% horse serum in PBS; antibody #2211, Cell Signaling Technology) overnight at 4° Celsius. Each slide was then washed and incubated with VectaStain universal secondary antibody (1:50 in 5% horse serum in PBS; Vector Laboratories). Slides were then subjected to R.T.U. VectaStain ABC reagent (Vector Laboratories, Inc. Bulingame, CA). Finally, sections were incubated with 3, 3’-diaminobenzidine (DAB) substrate (DAB Peroxidase (HRP) Substrate; Vector Laboratories, Inc. Bulingame, CA); counterstained with Vector Hematoxylin QS (H-3404, Vector Laboratories, Inc. Bulingame, CA); dehydrated; and cover slipped. Light microscopy was performed and images were acquired using a 20x objective on a Zeiss Axio Imager M2 imaging system. Images were then analyzed with ImageJ.