Protein:
Brain(Rat) lysate at 30ug;
Colon(Rat) lysates, 30ug;
Primary: Anti-ER-alpha/beta (SL0174R) at 1:200
Secondary: HRP conjugated Goat-Anti-Rabbit IgG(bse-0295G) at 1: 3000
Predicted band size : 58kD,66kD
Observed band size : 58kD
ER-alpha: 66kD; ER-beta: 58kD;
Paraformaldehyde-fixed, paraffin embedded (rat uterus); 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 (Estrogen Receptor alpha + beta) Polyclonal Antibody, Unconjugated (SL0174R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Tissue/cell: human endometrium tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min;
Incubation: Anti-ER-alpha/beta Polyclonal Antibody, Unconjugated(SL0174R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Tissue/cell: HUVEC cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (Estrogen Receptor alpha + beta
) Polyclonal Antibody, Unconjugated (SL0174R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody (SL0295G-FITC) at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Blank control (blue line): MCF7 (blue).
Primary Antibody (green line): Rabbit Anti-Estrogen Receptor alpha + beta antibody (SL0174R)
Dilution: 5μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC
Dilution: 1μg /test.
Protocol
The cells were fixed with 80% ethanol (Overnight at 4℃) and then permeabilized with 90% ice-cold methanol for 30 min on ice. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
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