Donkey anti mouse igg hrp

Individual dissected posterior eyecups were lysed in 160 μL of HNTG detergent buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl₂, 1% Triton X-100) freshly supplemented with 1% protease and phosphatase inhibitor cocktails (#P8340, Sigma-Millipore, and #78420, Thermofisher, respectively). Cleared lysates representing equal eyecup tissue fractions were separated on 12% SDS polyacrylamide gels using a standard Tris–glycine buffer system (Novex gels and solutions, Thermofisher). Proteins were transferred to nitrocellulose membranes (#88018, Thermofisher) for immunodetection with primary and appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. The primary antibodies used were porin (#4866, Cell Signaling, 1:5,000), RhoA (#sc-179, Santa Cruz Biotechnologies, Santa Cruz, CA, USA, 1:200), rhodopsin (clone B6-30, 0.01 μg/mL), and α-tubulin (#9099, Cell Signaling, 1:5,000). The secondary antibodies used were donkey-anti-rabbit IgG-HRP (#16029, Thermofisher, 1:10,000) and donkey-anti-mouse IgG-HRP (#16011, Thermofisher, 1:5,000). Chemiluminescence reaction signals from ECL-plus substrate (#NEL105001EA, Perkin-Elmer, Shelton, CT, USA) were captured with X-ray films (#3018, Denville Scientific, Swedesboro, NJ, USA), which were scanned and quantified by densitometry using Image Quant^TM TL 7.0 (GE Healthcare, Chicago, IL, USA).

The antibodies used in this study were obtained from the following sources: anti-survivin (71G4B7, Cat#2808), anti-XIAP (3B6, Cat#2045), anti-phospho-JAK1 (Y1034/1035; Cat#3331), anti-phospho-STAT3 (Y705; D3A7, Cat#9145), anti-cleaved PARP (Cat#5625T), anti-cleaved caspase 3 (Cat#9664) and anti-phospho-JAK2 (Y1007/1008; Cat#3771) from Cell Signaling Technology Inc. (Beverly, MA, USA); anti-actin (I-19, Cat#sc-1616), anti-GAPDH (0411, Cat#sc-47724), anti-JAK1 (B-3, Cat#376996), anti-phospho-STAT3 (Y705; B-7, Cat#sc-8059), anti-STAT3 (F-2, Cat#sc-8019), from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); anti-vinculin (Cat#V-4505) from Sigma-Aldrich. Antibody binding was detected with donkey anti-mouse IgG-HRP (Cat#A16017), donkey anti-rabbit IgG-HRP (Cat#A16029) and donkey anti-goat IgG-HRP (Cat#A15999) from Thermo Fisher Scientific Inc. (Waltham, MA, USA); Alexa Fluor™ 488-conjugated donkey anti-mouse (Cat#A31572, Molecular Probes, Eugene, OR, USA) was used for antibody binding detection in immunofluorescence assays.

Immunohistochemistry was employed to evaluate CSA expression in placenta tissues. After being deparaffinized and rehydrated, slides were treated by antigen retrieval buffer. Then the tissue sections were incubated with BSA for 1 h at room temperature. Then the sections were incubated with mouse monoclonal anti-chondroitin sulfate antibody (Abcam, Cambridge, United Kingdom) or control IgG (Abcam) at 4°C overnight. After being washed, the sections were incubated with HRP-donkey anti-mouse IgG (ThermoFisher, Waltham, United States). The sections were observed under a light microscope.