VALIDATION IMAGES
Paraformaldehyde-fixed, paraffin embedded Mouse brain; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with GPR30 Polyclonal Antibody, Unconjugated (bs-20643R) at 1:400 overnight at 4°C, DAB staining.
Paraformaldehyde-fixed, paraffin embedded Mouse brain; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with GPR30 Polyclonal Antibody, Unconjugated (bs-20643R) at 1:200 overnight at 4°C, DAB staining.
Paraformaldehyde-fixed, paraffin embedded Rat brain; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with GPR30 Polyclonal Antibody, Unconjugated (bs-20643R) at 1:200 overnight at 4°C, DAB staining.
Lane 1: Mouse Cerebrum lysates; Lane 2: Rat Cerebrum lysates; Lane 3: Human HepG2 cell lysates; Lane 4: Human SH-SY5Y cell lysates; Lane 5: Human MCF-7 cell lysates; Lane 6: Mouse Heart lysates; Lane 7: Rat Heart lysates probed with GPR30, Polyclonal Antibody, Unconjugated (bs-20643R) at 1:1000 dilution and 4˚C overnight incubation. Followed by conjugated secondary antibody incubation at 1:20000 for 60 min at 37˚C.
Tissue/cell:SH-SY5Y cell; 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 (GPR30) polyclonal Antibody, Unconjugated (bs-20643R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Paraformaldehyde-fixed, paraffin embedded (rat placenta); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Incubation with (GPR30) Polyclonal Antibody, Unconjugated (bs-20643R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
25 ug total protein per lane of various lysates (see on figure) probed with GPR30 polyclonal antibody, unconjugated (bs-20643R) at 1:1000 dilution and 4°C overnight incubation. Followed by conjugated secondary antibody incubation at r.t. for 60 min.