As a secondary antibody, we used FITC-conjugated polyclonal goat Fab fragments directed to mouse and RITCconjugated polyclonal goat Fab fragments directed to rabbit immunoglobulins (1: 100; Bioss
After washing with PBS, the sections were incubated overnight with polyclonal rabbit antibody GHR (1:80, bs-0654R, BIOSS
, Beijing, China) at 4[degrees]C.
Echinops griffithianus Bioss
. Centaurea iberica Trev.
Primary polyclonal antibodies of rabbit anti-rabbit collagen type I (1:20, Bioss
Bio., China), rabbit anti-rabbit MMP-2 (1:50, Boster Bio., China), and rabbit anti-rabbit MMP-9 (1: 20, Bioss
Bio., China) were applied overnight at 4[degrees]C.
Rabbit anti-[beta]-actin primary antibody was purchased from Bioss
Diabetic and control mice were randomly assigned to three groups in which different topical treatments were applied to the wounds (the saline group (C), the rabbit IgG isotype (Bioss
, Beijing, China) group (I), and the anti-RAGE antibody (Abcam, UK) group (R)).
The slides were incubated with polyclonal anti-A[beta]1-40 (1:200, Bioss
bs-0877R); anti-p-Tau (ser404) (1:200, Bioss
bs-2392R); and anti-4-HNE (1:800, Abcam: ab46545) antibodies individually overnight at 4[degrees]C.
Antibodies and the used were p53 (1: 2000, Upstate), Hdm2/MDM2 (1: 1000, Bioss
), p14/Arf (1: 1000, Cell Signaling), p21 (1: 2000, Millipore), [beta]-actin (1:5000, Sigma-Aldrich), Bax (1:1000, Cell Signaling), caspase-9 (1: 1000, Cell Signaling), caspase-3 (1: 1000, Cell Signaling), cleavage caspase-3 (1: 1000, Cell Signaling), mTOR (1: 10,000, Abcam), PUMA (1: 1000), NOXA (1: 1000), Bcl-2 (1: 1000), Beclin-1 (1: 1000, Cell Signaling), LC3 (1:2000, Abcam), anti-mouse IgG (H + L) horseradish peroxidase conjugate, and anti-rabbit IgG (H + L) horseradish peroxidase conjugate (1 : 3000, Bio-Rad).
Pancreatic and duodenal homeobox-1 (PDX1, bs-0923R) antibody was from Bioss
Immunoblotting was performed by incubating the PVDF membrane in TBS buffer containing 0.1% Tween and 5% nonfat milk for one hour at 4[degrees]C, followed by overnight incubation at 4[degrees]C with rabbit anti-survivin polyclonal antibody (Bioss
Inc., MA, USA) and mouse anti-IGF-I monoclonal antibody (Novus Biologicals, LLC, Littleton, CO, USA) at a dilution of 1: 1500.
Membranes were blocked and incubated with a set of primary antibodies (anti-p-IKKa, anti-IKKa/[sz], anti-I?Ba, and anti-p-I?Ba, Bioss
Inc., USA) overnight at 4[degrees]C.