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[IF=5.714] Jian-Lin Zhou. et al. CircSPI1_005 ameliorates osteoarthritis by sponging miR-370-3p to regulate the expression of MAP3K9. INT IMMUNOPHARMACOL. 2022 Sep;110:109064  WB ;  Mouse.  

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Sample:
Lymph node (Mouse) Lysate at 40 ug
Primary: Anti-Ki-67 (bs-23102R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 358 kD
Observed band size: 358 kD

Sample:
NIH/3T3 Cell Lysate at 30 ug
Primary: Anti-Ki-67 (bs-23102R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 358 kD
Observed band size: 358 kD

Tissue/cell: Hela 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 (Ki-67) polyclonal Antibody, Unconjugated (bs-23102R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG-AF488 antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Blank control:HL-60.
Primary Antibody (green line): Rabbit Anti-Ki-67 antibody (bs-23102R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF488
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 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(blue): HepG2(fixed with 2% paraformaldehyde for 10 min at 37℃).
Primary Antibody:Rabbit Anti-Ki-67 antibody (bs-23102R,Green); Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA;
Isotype Control Antibody: Rabbit IgG(orange) ,used under the same conditions;
Secondary Antibody: Goat anti-rabbit IgG-FITC(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.