Protein block serum free ready to use solution

Immunohistochemistry was performed as described previously [16] (link). In brief, 3-µm-thick sections of the aorta, fixed in 4% paraformaldehyde, were deparaffinized with xylene and graded alcohol from 100% to 70% and autoclaved at 121°C for 15 min in 10 mmol/l citrate buffer (pH 6.0). Following this, section slides were immersed in 3% H₂O₂ for 20 min to block endogenous peroxidase activity, followed by 10 min in Protein Block Serum-Free Ready-to-Use solution (DakoCytomation Japan, Kyoto, Japan) to reduce the non-specific background. The slides reacted with specific polyclonal antibodies against CD3 (1∶100, ab5690; Abcam, Cambridge, UK), CD31 (ab28365; Abcam), interleukin (IL)-6 (1∶400, ab6672; Abcam), and regulated on activation, normal T cell expressed and secreted (RANTES) (1∶50, NBP1-19769, Novus, Littleton, CO, USA) or monoclonal antibodies against F4/80 (1∶10,000, CL8940AP, Cedarlane, Hornby, ON, Canada) at 4°C overnight. Following this, they reacted with peroxidase-based EnVision+ (DakoCytomation Japan) for 30 min at room temperature, and were developed in 0.05% 3,3′-diaminobenzidine containing hydrogen peroxide. All sections were counterstained with hematoxylin and eosin. A catalyzed signal amplification system (CSA-Dako–Cytomation) was used to enhance the detection of F4/80 antigen, according the to manufacturer's instructions.

HaCaT cells (2 × 10⁴ cells/well) were seeded in a four-well chamber and cultured for 18 h. After the medium was replaced with a cytokine mixed medium containing TNF-α (20 ng/mL; PeproTech, Rocky Hill, NJ, USA) and IL-6 (10 ng/mL; PeproTech), the cells were incubated for 24 h, fixed using 5% neutral-buffered formalin for 10 min, and permeabilized using 0.2% Triton X-100 (Sigma-Aldrich Co.) in PBS for 10 min. The cells were then blocked using Protein Block Serum-Free Ready-To-Use solution (Dako North America, Inc.) at 25 °C for 1 h and incubated with LPAR1 antibody (ab23698; Abcam) diluted at 1:200 in Antibody Diluent (Dako North America, Inc.) at 4 °C overnight. Then, the cells were incubated with anti-Rabbit Alexa 488 secondary antibody (Abcam) diluted at 1:200 in Antibody Diluent. The nuclei were stained with DAPI (Invitrogen) diluted at 1:1000 in PBS, mounted with fluorescence mounting media (Dako North America, Inc.), and observed using a confocal microscope (Carl Zeiss Inc.).

B16F10 cells (2 × 10⁴ cells/well) were seeded overnight in the Nunc™ Lab-Tek™ II Chamber Slide™ system (Thermo Fisher Scientific, Rochester, NY, USA). The cells were pretreated with vehicle (DMSO) or 10 μM SB 203580 for 1 h and then further treated with or without a final concentration of 10 μg/mL of PME for 24 h. The cells were washed twice with DPBS and then fixed in 4% paraformaldehyde for 15 min at 25°C. After another wash with DPBS, the cells were blocked in Protein Block Serum-Free Ready-to-Use solution (Dako North America, Inc., Carpinteria, CA, USA) at 25°C for 1 h, followed by incubation overnight with anti-tyrosinase (Cell Signaling Technology) antibody diluted in Antibody Diluent (1 : 200, Dako North America, Inc.) at 4°C. The cells were then stained with Alexa Fluor 546-conjugated secondary antibody (1 : 200, Life Technologies, Carlsbad, CA, USA) at 25°C for 2 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, San Diego, CA, USA) diluted in DPBS (1 : 1000) at 25°C for 5 min. Cells were mounted with Fluorescence Mounting Medium (Dako North America, Inc.) and then observed under a confocal microscope (LSM700; Carl Zeiss Inc., Oberkochen, Germany).