[IF=0.88] Erikci, Acelya, Gulberk Ucar, and Samiye Yabanoglu-Ciftci. "Role of serotonin in the regulation of renal proximal tubular epithelial cells." Renal failure 38.7 (2016): 1141-1150.  IF(ICC) ;  Mouse.  

PubMed:27277500

Tissue/cell: mouse kidney 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-CD13/APN/ANPEN Polyclonal Antibody, Unconjugated(bs-1383R) 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-CD13/APN/ANPEN Polyclonal Antibody, Unconjugated(bs-1383R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Black line : Positive blank control U937); Negative blank control (A431）
Green line : Primary Antibody (Rabbit Anti- CD13 antibody (bs-1383R) )
Orange line：Isotype Control Antibody (Rabbit IgG) .
Blue line : Secondary Antibody (Goat anti-rabbit IgG-AF647)
U937（Positive）and A431 Negative control）cells (black) were incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with CD13 Antibody(bs-1383R)at 1:100 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).

Blank control: U937 (blue).
Primary Antibody:Rabbit Anti- CD13 antibody(bs-1383R), 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-1383R, 1μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 1 0% 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.