Peroxidase conjugated affinipure goat anti mouse igg h l

The tissue sections were examined by H&E staining for histological verification of disease status. Sections were dewaxed in xylene and rehydrated in fractionated ethanol. Heat-induced antigen retrieval was performed for epitope unmasking in 10 mM citric acid buffer (pH 6). Slides were blocked with 2.5% horse serum at room temperature (RT) for 1 h and incubated with primary antibody overnight in a humidified chamber at 4 °C. Next, the slices were incubated overnight at 4 °C in primary antibody solution (anti-AURKB, 1:500, ab45145; Abcam, Cambridge, MA, USA). After repeated washing, the slices were incubated with the secondary antibody (peroxidase-conjugated Affinipure goat anti-mouse IgG (H + L), 1:200 dilution, ZB-2305, ZSGB-BIO, Beijing, China) for 1 h at room temperature. The slides were then incubated with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin. Image acquisition was performed with an Olympus IX71 fluorescence microscope.

Cells were lyzed with a lysis buffer containing phenylmethyl sulfonylfluoride (PMSF) (Beyotime, Shanghai, China) at 4°C. Proteins were quantified using a BCA protein assay kit (ComWin Biotech, Beijing, China). Protein lysates (50 μg) were separated by 10% SDS-PAGE gels (Invitrogen) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Beyotime, Shanghai, China). The membranes were blocked with 5% non-fat dry milk in Tris-phosphate buffer containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then treated with the primary antibodies at 4°C overnight. After washing with TBST, the membranes were incubated with peroxidase-conjugated affinipure goat anti-rabbit IgG (H+L) (ZSGB-BIO, 1:5000, ZB-2301) and peroxidase-conjugated affinipure goat anti-mouse IgG (H + L) (ZSGB-BIO, 1:5000, ZB-2305) for 1 h at room temperature. The blots were visualized using an ECL kit (ComWin Biotech, Beijing, China), and quantified using the Image J Software, normalized to β-actin. The primary antibodies were: E-cadherin (1:500, Cell signaling, #3195), Vimentin (1:1000, Cell signaling, #5741), ERK1/2 (1:1000, Cell signaling; #4695), p-ERK1/2 (Thr202/Tyr204) (1:1000, Cell signaling, #9101), α-catenin (1:500, Proteintech, Catalog number: 66221-1-Ig), β-catenin (1:1000, Santa Cruz, sc-7963), and β-actin (1:1000, Bioss).

Azithromycin and N-acetylcysteine (NAC) were obtained from National Institutes for Food and Drug Control (Beijing, China). Azithromycin was dissolved in anhydrous ethanol; NAC was dissolved in distilled water. Anti-caspase-3, anti-Akt (pan), anti-p44/42 MAPK (Erk 1/2), anti-p38 MAPK, anti-PARP and p62 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3B antibody was purchased from Sigma (St. Louis, MO, USA). Azide-free anti-human CD261 (DR4) antibody was obtained from Diaclone (Besancon, France). Anti-DR5 antibody was obtained from ProSci (San Diego, California, USA). β-Actin (6G3) and GAPDH (1C4) monoclonal antibodies were purchased from AmeriBiopharma (Wilmington, Delaware, USA). Secondary antibodies included peroxidase-conjugated affiniPure goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) (ZSGB-BIO, Beijing, China). Caspase inhibitor zVAD-fmk and RIP1 inhibitor necrostatin-1 were purchased from Selleck.cn (Houston, TX, USA). Chloroquine (CQ), acridine orange hemi (zinc chloride) salt (AO) and sulforhodamine B (SRB) were from Sigma.