Fitc goat anti rabbit igg

PRP diluted in PBS was incubated with CD41a (anti-human, APC, Invitrogen) and TRPM8 antibody (1:250 dilution, #ACC-049, Alomone) for 30 minutes at room temperature, and followed by the incubation with the secondary antibody (FITC- goat anti-rabbit IgG, 1:250 dilution, #L43001 Invitrogen) for 30 minutes at room temperature. The samples were fixed with PFA (final concentration 1%).
Samples were run on the LSR II flow cytometer (BD Biosciences). For each sample, a total of 100,000 events were acquired. Data were analyzed by FlowJo V10 software. Platelets were distinguished from other blood cells by using forward (FSC) and side scatter (SSC) and CD61 positive events. The gating strategy for PAC-1 and P-selectin was selected based on the binding of the appropriate isotype antibodies.

The cross-section samples of the aortic sinus were used for immunofluorescence. The slides were blocked in 10% goat serum diluted with PBS for 30 min and incubated overnight at 4 degrees Celsius with various primary antibodies: LC3 (L7543, Sigma, USA), p62 (PM045, MBL, Japan), CD11b (553312, BD, USA), TFEB (13372-1-ap, proteintech, China), LAMP1 (EPR21026, abcam, UK). After rewarming at room temperature for 10 min, the sections then were incubated with the relevant secondary antibody for another 1 h. The secondary antibodies used were FITC goat anti-rabbit IgG (Invitrogen, USA), TRITC goat anti-rabbit IgG (Invitrogen, USA), FITC goat anti-rat IgG (Invitrogen, USA). The nuclei were stained with 0.5 g/L DAPI for 10 min. Images were obtained using a Confocal microscope (ZEISS LSM 800) and the images were analyzed with Image Pro Plus 6.0.
Cells were seeded onto glass slides. After the indicated treatments, cells were fixed with 4% paraformaldehyde and permeabilized by 0.1% Triton X-100. Subcequently, cells were incubated with primary antibodies and secondary antibody as described in histochemistry, cells fluorescence intensity was observed under Confocal microscope (ZEISS LSM 800) and representative cells were selected and photographed. Quantification analysis was performed using ImageJ.

Berberine (BBR) was obtained from CDMUST Biotech (Chengdu, China). Lipopolysaccharide (LPS) (E. coli) was from Biosharp (Beijing, China). Compound C (CC) was purchased from Calbiochem (Darmstadt, Germany). AG490 was from Sigma (Darmstadt, Germany), S3I-201 and ML385 were purchased from Selleck (CA, USA). Antibodies against phospho-AMPKα1 (T172) (ab133448) and AMPKα1 (ab32047) were from Abcam (MA, USA), those against STAT3 (79D7), phospho-STAT3 (Y705) (D3A7), were from Cell Signaling Technology (BSN, USA), those against phospho-JAK2 (Y1007+Y1008) (CY6570) was from Abways Technology (Shanghai, China), phospho-Nrf2 was from Bioss Antibodies (Beijing, China). FITC-goat anti-rabbit IgG was from Invitrogen (CA, USA). Plasmids transfection reagent was Jet PRIME from PolyPlus (Illkirch, France).