Immunocytochemistry of NFκB, Iba-1, CD11b, MMP-13, CD68

After treatment was stopped, cells were prepared for immunocytochemistry by washing with phosphate buffered saline (PBS) for 5 min, followed by a 20-min fixation with PBS containing 4% (w/v) paraformaldehyde and 4% (w/v) sucrose, pH 7.4 at room temperature. Cells were then permeabilized in PBS containing 0.1% (v/v) Triton X-100 for 5 min, and blocked for 1 h with PBS containing 10% (v/v) goat serum followed by overnight incubation at 4°C with rabbit anti-NFκB (1:1000; α-p65; Abcam), rabbit Iba-1 (1:750; Wako), rat CD11b (1:1000; EBT), rabbit MMP-13 (Abcam; 1:100), or rat CD68 (1:400; Bio-Rad Laboratories) in blocking buffer containing 10% goat serum. Antibody:antigen complexes were visualized following incubation with Alexa Fluor 594 or 488 conjugated goat IgG secondary antibody (1:1000) in PBS containing 0.1% (v/v) triton X-100 and 1% goat serum and subsequently counterstained with 4',6-diamidino-2-phenylindole (DAPI; 13.0 ng/μL) in PBS, followed by two 5 min PBS washes. Coverslips were mounted with Hydromount and cells were imaged and captured using a Zeiss Axioskop fluorescent microscope and AxioCam HRm camera (Carl Zeiss) (Béraud et al., 2011 (link), 2013 (link); Béraud and Maguire-Zeiss, 2012 (link); Daniele et al., 2014 (link), 2015 (link)). Images and subsequent analyses were completed by an observer blinded to treatment.