Cyr61 antibody

RPMI 1640 medium, Dulbecco’s modified Eagle medium, trypsin/EDTA, and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 5-FU, ADR, tamoxifen (TAM), PAC, and quercetin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Docetaxel (DOC) was purchased from Dong-A Pharmaceutical (Seoul, Korea). MRP1, p65, poly (ADP-ribose) polymerase (PARP), β-actin, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L), and HRP-conjugated goat anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). CYR61 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Cells were plated for immunofluorescence (IF) onto 24 well plate circular glass inserts and fixed upon reaching 70% confluence. Fixation was performed by incubating cells with 4% Paraformaldehyde for 10 minutes followed by a two minute fixation with ice cold pure methanol. The cells were blocked with 2% horse serum diluted in 1× PBS for 1 hour, then incubated with the primary CYR61 antibody (Santa Cruz Biotechnology, Rabbit pAB 1∶400 dilution) for 1 hour. The cells were washed 3 times with 1× PBS for 5 minutes each. The cells were then incubated with secondary FITC fluorescence antibody (Santa Cruz Biotechnology, 1∶500) for one hour. Subsequently, the cells were washed 3 times with 1× PBS for 5 minutes each time, and mounted with VECTASHIELD Mounting Media with DAPI (Vector Laboratories). The cells were then imaged; FITC representing the Cyr61 protein is green, and DAPI representing the nuclear counterstain is blue.

The IHC staining was done as previously described [24 (link)]. Mouse on mouse blocking reagent for vimentin staining, ABC-HRP kit and ABC-AP kit were purchased from Vector Laboratories (Burlingame, CA). CD31 antibody (Thermo Fisher Scientific, Reinach, Switzerland) and biotinylated goat anti-rabbit secondary antibody (Vector Laboratories) were diluted 1:50 and 1:200, respectively. Vimentin antibody and biotinylated goat anti-mouse secondary antibody (Vector Laboratories) were diluted 1:200 and 1:800, respectively. CYR61 antibody (Santa Cruz Biotechnology, Inc., Heidelberg, Germany, #sc13100) and biotinylated goat anti-rabbit secondary antibody were diluted 1:150 and 1:600, respectively. For CD31/vimentin double-staining, tissues were first stained with anti-CD31 and biotinylated goat anti-rabbit secondary antibody, followed by anti-vimentin-staining and biotinylated goat anti-mouse secondary antibody. Substrates used were DAB (Sigma-Aldrich) and Vector Red (Vector Laboratory).