[IF=1.813] Yang Wanget al. Electroacupuncture Improves Blood Pressure in SHRs by Regulating the Immune Balance between Th17 and Treg. Evid Based Complement Alternat Med . 2020 Jul 7;2020:5375981.  WB ;  rat.  

PubMed:32724324

Sample:
Liver (Mouse) Lysate at 40 ug
Primary: Anti-ROR Gamma (bs-3863R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 57 kD
Observed band size: 50 kD

Sample:
Lane1: Mouse Muscle Lysates
Lane2: Rat Muscle Lysates
Primary: Anti-ROR Gamma (bs-3863R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 57 kD
Observed band size: 50 kD

Sample: Liver (Mouse) Lysate at 40 ug
Primary: Anti- ROR Gamma (bs-3863R) at 1/300 dilution
Secondary: HRP conjugated Goat-Anti-rabbit IgG (bs-0295G-HRP) at 1/5000 dilution
Predicted band size: 57 kD
Observed band size: 57 kD

Paraformaldehyde-fixed, paraffin embedded (mouse intestines); 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 (Gamma) Polyclonal Antibody, Unconjugated (bs-3863R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

Tissue/cell: mouse colon 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,C-0005) at 37℃ for 20 min;
Incubation: Anti-ROR Gamma Polyclonal Antibody, Unconjugated(bs-3863R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: mouse colon 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,C-0005) at 37℃ for 20 min;
Incubation: Anti-ROR Gamma Polyclonal Antibody, Unconjugated(bs-3863R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

HepG2 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 (ROR Gamma) polyclonal Antibody, Unconjugated (bs-3863R) 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:Molt-4.
Primary Antibody (green line): Rabbit Anti-ROR Gamma antibody (bs-3863R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF647
Dilution: 1μg /test.
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 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.

Blank control（black line）:HepG2.
Primary Antibody (green line): Rabbit Anti-ROR Gamma antibody (bs-3863R)
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.