Goat anti rat alexafluor555

All cell types were fixed with 4% paraformaldehyde (45 min) and treated with blocking buffer for 45 min. Only fixed nRCMs were treated with permeabilisation buffer (3% Triton X-100) for 10 minutes preceding blocking procedure. After blocking, nRCMs were incubated for 3 hours at 37 °C with the primary antibodies mouse anti-α-Actinin (1:1000, Sigma), and rat anti-α-Tubulin (1:1500, Serotec). NRCFs were treated with rabbit anti-Collagen 1α1 (1:500, Abcam) and rat anti-α-Tubulin (1:750) antibody overnight at 4 °C. BMDMs were treated with rabbit NFκB p65 antibody (1:300 Santa Cruz #sc-372) and rat anti-α-Tubulin (1:750) overnight at 4 °C. After primary antibody incubation, nRCMs and nRCFs were incubated with secondary antibodies goat anti-rat AlexaFluor555 (1:750, Life Technologies), goat anti-mouse AlexaFluor488 (1:500, Life Technologies) and Hoechst (1:6000, Invitrogen) for 1 hour at room temperature. BMDMs were incubated with secondary antibodies goat anti-rat AlexaFluor555 (1:500), goat anti-rabbit AlexaFluor488 (1:750, Life Technologies) and Hoechst for 1 hour at room temperature.