Goat anti mouse igg h l antibody

Fluorescein Diacetate (FDA), Calcein‐AM, Vybrant® DiI Cell‐Labeling Solution, and Geltrex™ LDEV‐Free Reduced Growth Factor Basement Membrane Matrix were purchased from Thermo Fisher Scientific Inc. Goat Anti‐Mouse IgG (H + L) Antibody and Rhodamine conjugate were from Sigma Aldrich. Keloid fibroblasts (KF110) were ordered from Cell Research Corporation (Singapore). Dulbecco's modified eagle medium (DMEM) (w/4.5 g/L d‐glucose, w/phenol red), fetal bovine serum (FBS), penicillin‐streptomycin (10,000 U/ml), Phosphate buffered saline (1x, PBS), and trypsin‐EDTA (0.25%, 10x) were obtained from Gibco Life Technologies (USA). All reagents were of analytical reagent grade and used without further purification.

For protein expression analyses with western blots, PBL were first lysed in lysis buffer (9 M Urea, 2 M Thiourea, 65 mM Dithioerythritol, 4% CHAPS). Proteins were then separated by SDS-PAGE on 8% gels (7 μg protein/slot) and blotted semidry onto 8.5 × 6 cm PVDF membranes (GE Healthcare, Freiburg, Germany) and blocked with 4% BSA (1 h). Blots were incubated overnight with respective primary antibodies: rabbit anti-pSTAT1 Tyr701, rabbit anti-pSTAT3 Tyr705 (Cell Signaling, Darmstadt, Germany, 1:500) or mouse anti-beta actin (Sigma, Taufkirchen, Germany, 1:5000). As secondary antibodies, either HRP-coupled goat anti-rabbit IgG (H+L) antibody (Cell Signaling, Darmstadt, Germany, 1:5000) or goat anti-mouse IgG (H+L) antibody (Sigma-Aldrich, Taufkirchen, Germany, 1:5000) were used (1 h). Signals were detected by enhanced chemiluminescence on X-ray film (SUPER-2000G ortho, Fuji; Christiansen, Planegg, Germany). Films were scanned on a transmission scanner and densitometric quantification of Western blot signals was performed using ImageJ software (open source: http://imagej.nih.gov/ij/). Abundances of pSTAT1 and pSTAT3 were subsequently normalized to beta actin.