Alexa fluor594 antibody

H9C2 cells were fixed in 4% paraformaldehyde (PFA) for 10 min and permeabilized using 0.2% Triton X-100. The cells were blocked using 10% bovine serum albumin (BSA) for 1 h and then incubated with anti-RUNX1 (1:200, Abcam) primary antibody at 4°C overnight. Further, the cells were washed with PBS 0.05% Tween (PBST) followed by incubation with Alexa Fluor594 antibody (1:1000, Abcam). After 1 h of incubation at 37°C, cell nuclei were stained with DAPI. Images were obtained by fluorescence microscope (Leica AF6000). The quantification of RUNX1 positive cells was performed using the ImageJ software.
The heart was dissected and then fixed in 4% PFA for 4 h. The heart sections (5 µm) were embedded in polyethylene glycol. USP7 (1:100; Cell Signaling Technology) and p53 (1:500; Cell Signaling Technology) antibodies were incubated to evaluate the interaction of USP7 and p53 in I/R rats. The sections were then incubated with Alexa Fluor 488 antibody (1:1000; Abcam) or Alexa Fluor594 antibody (1:1000, Abcam) for 2 h at 37°C. The nuclei were stained with DAPI. The images were visualized using Aconfocal microscopy (Zeiss 710).

Cells were seeded on poly‐l‐lysine‐coated coverslips of 12‐well plates and transfected with GPCRs and MRAP1/MRAP2‐Flag‐F2. After 24 h transfection, cells were fixed with 4% PFA for 20 min at room temperature. Samples were then incubated with anti‐FLAG antibody (Cell Signaling) at 1:5000 ratio to detect surface expression of MRAPs. After washing three times, samples were incubated with Alexa Fluor594 antibody (Abcam) at 1:5000 ratio. Finally, cells were sealed with ProLong (R) Gold Antifade containing DAPI Molecular Probes (Cell Signaling). Images were captured under 60× oil objective with Zeiss confocal microscopy (LSM880). Each YFP group was repeated at least twice, with three fields of view selected.