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PubMed:30075109

Paraformaldehyde-fixed, paraffin embedded (Human brain glioma); Antigen retrieval by microwave in sodium citrate buffer (pH6.0) ; Block endogenous peroxidase by 3% hydrogen peroxide for 30 minutes; Blocking buffer (3% BSA) at RT for 30min; Antibody incubation with (phospho-GEF H1 (Ser886)) Polyclonal Antibody, Unconjugated (bs-2201R) at 1:400 overnight at 4℃, followed by conjugation to the secondary antibody (labeled with HRP)and DAB staining.

A431cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (phospho-GEF H1 (Ser886)) polyclonal Antibody, Unconjugated (bs-2201R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Blank control（black line）:RAW264.7. Primary Antibody (green line): Rabbit Anti-phospho-GEF H1 (Ser886) antibody (bs-2201R) Dilution:1ug/Test; Secondary Antibody（white blue line）: Goat anti-rabbit IgG-AF488 Dilution: 0.5ug/Test. Isotype control（orange line）: Normal Rabbit IgG Protocol The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃, The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.