Lsm 780 upright confocal microscope 63x objective

To visualize TRPM8 expression in IPA and VSMCs, confocal microscopy was performed. Arteries were embedded in optimal cutting temperature medium and frozen in liquid nitrogen. Slides containing transverse cross-sections (10 μm) of frozen arteries were immersed into acetone/methanol solution (1:3 proportion) for 10min, washed by physiological buffer solution (PBS, 1X) and blocked with hydrogen peroxide (3%) for 30 min. In the case of the experiments performed with VCMCs, they were fixed in 4% paraformaldehyde (Thermo Fisher Scientific) for 10 min. Cross-sections/cells were blocked in 1X PBS with 0.01% Triton X-100 (Thermo Fisher Scientific) and 5% horse serum. The slides were then incubated with anti-TRPM8 (1:30; Abcam/ab3243) primary antibody overnight. Next, slides were incubated with goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody Alexa Fluor 594 (1:500) in 1X PBS and 5% BSA for 60 min. Vectashield HardSet Antifade Mounting Medium with DAPI (H-1500) (Vector Laboratories, Inc., Burlingame, CA) was then applied to slides with coverslips. IPA or VSMCs were visualized using a Zeiss LSM 780 Upright confocal microscope (63X objective) (Carl Zeiss MicroImaging, Oberkochen, Germany).

To visualize F actin bundles and α-tubulin, VSMCs were grown in Lab-Tek II Chamber Slide w/Covers (Thermofisher), treated with non-formylated peptides or F-MIT (20 min, 10 μM), and confocal microscopy was performed. After treatment, VSMCs were fixed in 4% paraformaldehyde (Thermofisher) for 10 min and blocked in 1X PBS with 0.01% Triton X-100 (Thermofisher) and 5% horse serum. The slides were then incubated with mouse anti-α-tubulin (1:100; Sigma) primary antibody in 1X PBS and 5% bovine serum albumin (BSA) overnight. Next, slides were incubated with goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor 488 (1:200; Thermofisher /A-11008) and rhodamine phalloidin (1:40; Thermofisher /R415) in 1X PBS and 5% BSA for 90 min. Vectashield HardSet Antifade Mounting Medium with DAPI (H-1500) (Vector Laboratories, Inc., Burlingame, CA) was then applied to slides with coverslips. Vascular smooth muscle cells were visualized using a Zeiss LSM 780 Upright confocal microscope (63X objective) (Carl Zeiss MicroImaging, Oberkochen, Germany).