Rabbit anti p akt1 2 3 ser473 polyclonal antibody sc 101629

Immunohistochemistry was performed using an SABC immunohistochemistry kit (Boster Bioengineering Co., Wuhan, China). Formalin-fixed, paraffin-embedded eye tissue sections (6-µm-thick) were placed on slides, deparaffinized in xylene and rehydrated by incubation in graded ethanol baths in PBS. Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol. The sections were then treated with 10% normal goat serum and incubated overnight with the following commercially available primary antibodies: rabbit anti-Cyr61 polyclonal antibody (ab24448; Abcam), mouse anti-p-PI3K monoclonal antibody (sc-12929), or rabbit anti-p-AKT1/2/3 (Ser473) polyclonal antibody (sc-101629) (both from Santa Cruz Biotechnology, Inc.) at 4°C. The sections were incubated with biotinylated horse secondary antibody againts mouse IgG (ZB-2020; Zhongshan Jinqiao Biotechnology Co., Ltd.) and reacted with the avidin-biotinylated peroxidase complex. The primary antibody was replaced with PBS for the negative controls, and 3,3′-diaminobenzidine (DAB) was used as the chromogen. The sections were counterstained with hematoxylin, dehydrated and mounted. Images were digitally captured using an Olympus B201 optical microscope (Olympus).

The HUVECs were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min. The cells were then washed with 0.1% Triton X-100 for 10 min and twice with PBS, and were incubated with 1% bovine serum albumin (BSA) at room temperature for 1 h. The cells were incubated overnight with the following commercially available primary antibodies: rabbit anti-Cyr61 polyclonal antibody (ab24448; Abcam, Cambridge, UK), mouse anti-p-PI3K monoclonal antibody (sc-12929), or rabbit anti-p-AKT1/2/3 (Ser473) polyclonal antibody (sc-101629) (both from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The cells were washed thrice with PBS. The primary antibodies were replaced by isotype controls, which were used as negative controls. The cells were then treated with fluorescence-conjugated secondary antibody [FITC-conjugated AffiniPure rabbit anti-goat IgG (H+L; ZF-0314), or tetramethylrhodamine (TRITC)]-conjugated AffiniPure goat anti-mouse IgG (H+L; ZF-0313) (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) for 1 h and counter-stained with 0.5 µg/ml 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Institute of Biotechnology, Jiangsu, China) for 5 min. Images were digitally captured using a confocal laser scanning microscope (FV1000; Olympus Corp., Tokyo, Japan).