Dylight 594 goat anti mouse igg

HK-2 cells were cultured in 6-well plate format for cell immunofluorescence analysis. After treatment of rosiglitazone (100 μM) and TGF-β (20 ng/ml) for 48 h, the medium was removed and cells were cleaned three times with PBS. Cells in coverslips of 6-well plate were fixed in 4% paraformaldehyde for 15 min, and then blocked with 10% goat serum (Boster, AR0009, China) for 1 h before incubation with primary antibodies against 4-HNE (1:100 dilution), Fibronectin (1:250 dilution) and α-SMA (1:250 dilution) at 4 °C for 24 h. After incubation, coverslips were rinsed three times with PBS for 5 min and incubated with the secondary antibodies Dylight 488 goat anti-rabbit IgG (Abbkine, California, USA) and Dylight 594 goat anti-mouse IgG (Abbkine, California, USA) for 60 min at room temperature. After antifade mounting medium with DAPI (Beyotime, P0131, China), samples were observed under confocal microscopy (Nikon C2, Japan) and fluorescence intensity was analyzed using ImageJ software.

The full-length sequence of MDV-1’s pp38 gene was synthesized and cloned into the eukaryotic expression plasmid pEGFP-N1 (SunYa, China) to generate the pEGFP-N1-pp38 plasmid, followed by transfection into 293T cells in 24-well plates using LipofectamineTM 2000 (Thermo Fisher Scientific, Basingstoke, UK) according to the manufacturer’s instructions. Two days later, the cells were fixed with precooled methanol/acetone (v/v = 1:1) and blocked with 5% skimmed milk, and then incubated with the positive supernatants of pp38 mAb candidates, followed by incubation with the secondary antibody DyLight 594 Goat Anti-Mouse IgG (Abbkine, USA). The pp38-specific mAb BD1 served as a positive control. Finally, the results were checked and recorded under a fluorescence microscope.

For immunofluorescent staining, brain samples were routinely collected, and 10-μm coronal frozen sections were made. The immunofluorescent staining procedure was performed as previously reported (19 (link)). The following primary antibodies were used: PDGFR-β (ab32570, Abcam), biotinylated lectin (B-1175, Vector Laboratories), fibrin (ogen) (ab34269, Abcam) and P-selectin (60322-1-Ig, Proteintech). Secondary antibodies, including AMCA-streptavidin (SA-5008, Vector Laboratories), DyLight 488 goat anti-rabbit IgG (A23220, Abbkine), and DyLight 594 goat anti-mouse IgG (A23410, Abbkine) were used. Three nonadjacent coronary sections from each brain sample with a minimum distance of 100 μm from one another were used. Five randomly selected visual fields per section were observed and analyzed by a blinded observer using Image-pro plus (IPP) 6.0 software by a blinded observer.