Anti lpar 1

The DuoLink In situ Red Starter kits mouse/rabbit and goat/mouse (Sigma-Aldrich, Merck, Darmstadt, Germany) were used according to the manufacturer’s protocol for Duolink In situ solutions (Sigma-Aldrich, Merck, Darmstadt, Germany). Anti-MRTF-A (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-FLNA (Millipore, Merck, Darmstadt, Germany), anti-LPAR-1 (Abcam, Cambridge, UK), and anti-LPAR-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies were used as primary antibodies. Data were visualized with a confocal microscope (Carl Zeiss, Oberkochen, Germany) or a fluorescence microscope (Motic, Wetzlar, Germany), and data were analyzed by 5–15 randomly selected fields.

O4 immunostaining was performed on live, unfixed cells. All other immunostaining was performed after cells were fixed with 4% paraformaldehyde. O4 monoclonal antibodies (1:20, grown from hybridoma cells in our lab; Knapp and Hauser, 1996 (link)) were applied at room temperature for 15 min. Goat anti-mouse IgM-Cy3 (1:1000, Millipore, Billerica, MA) was used to probe bound O4 antibodies. All LPA receptor antibodies (Anti- LPAR1 (Abcam), LPAR2, and LPAR3; Santa Cruz, Dallas, TX; LPAR4, Alomone labs, Hadassah Ein Kerem, Israel; and LPAR6, Acris, San Diego, CA) were applied at 1:1000 at room temperature for 1 hr, and corresponding secondary antibodies were conjugated to Alexa 488 (1:2000, Life Technologies). Antibody specific to ATX (Cosmo Bio, Japan) was also used at 1:1000 at room temperature. The cell nucleus was stained with Hoechst 33342 dye (1:2000, Life Technologies). Images were taken on a confocal microscope (Zeiss LSM 700, Carl Zeiss, Thornwood, NY) and processed using Zeiss Zen 2010 software. OLG morphology was assessed by determining the overall process network (total O4⁺ area minus the area occupied by the cell body) as described previously (Dennis et al., 2008 (link)), but using ImageJ software (NIH). Evaluators were blinded to treatment groups.