Alexa fluor 633 conjugated anti rabbit igg

MCF-7 cells seeded on chamber slides were treated with 25 or 50 μg/mL GSE for 2 h or 24 h, and the related controls were used for immunochemistry and confocal microscopy analyses as described in [32 (link)]. Different experiments were done using both anti-Cx43 from Sigma-Aldrich and from Zymed as primary antibody (1:250 in PBS-BSA). In addition, Alexa Fluor^® 633-conjugated anti-rabbit IgG or Alexa Fluor^® 488 conjugated anti-rabbit IgG as secondary antibodies, and PI or SYBRGreen (all products from Invitrogen) as nuclear counterstain were used. Only images obtained from immunodetection with anti-Cx43 from Sigma-Aldrich, Alexa Fluor^® 633-conjugated anti-rabbit IgG and SYBR green are shown. Negative controls were obtained with non-immune serum followed by a secondary antibody or with primary or secondary antibodies only. A minimum of five randomly selected areas from each chamber of each GSE treatment or control were analysed by confocal microscopy (LSM Pascal, Zeiss, Munich, Germany).

The following primary antibodies (anti-human) were used: AKT, phospho-AKT, MAPK(ERK1/2), phospho-MAPK(ERK1/2), Jak2, phospho-Jak2, Stat3, and phospho-Stat3 (Cell Signaling Technology, MA, USA), GAPDH (Multisciences, China), 4H7 (Soochow University-Bright Scistar, China), CD276 (R&D Systems, MN, USA), and CXCR4 (EMD Millipore, MA, USA).
The following secondary antibodies were used: horseradish peroxidase-conjugated rabbit anti-goat, goat anti-mouse, and anti-rabbit antibodies (Multisciences, China). Proteins were visualized with an ECL detection kit (Bio-Rad, CA, USA). The following fluorescent secondary antibodies were used: Cy3-conjugated anti-goat IgG (Invitrogen, CA, USA) and Alexa Fluor 633-conjugated anti-rabbit IgG (Invitrogen, CA, USA). The following antibodies were used for flow cytometry: B7-H3-APC (eBiosciences, CA, USA) and CXCR-4-PE-cy7 (eBiosciences, CA, USA).