[IF=5.719] Lingzi Feng. et al. A Closed-Loop Autologous Erythrocyte-Mediated Delivery Platform for Diabetic Nephropathy Therapy. NANOMATERIALS-BASEL. 2022 Jan;12(20):3556  FC ;  Rabbit.  

PubMed:36296745

Sample:
Lane 1: Hela (Human) Cell Lysate at 30 ug
Lane 2: Huvec (Human) Cell Lysate at 30 ug
Lane 3: A549 (Human) Cell Lysate at 30 ug
Primary: Anti-CD55 (bs-1552R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 35 kDa
Observed band size: 73 kDa

Tissue/cell: human lung carcinoma; 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-CD55 Polyclonal Antibody, Unconjugated(bs-1552R) 1:500, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Blank control: U937(blue).
Primary Antibody: Rabbit Anti- CD55 antibody(bs-1552R), 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-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min). Primary antibody (bs-1552R, 1μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 10% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.