[IF=1.679] Kim et al. Blocking junctional adhesion molecule C promotes the recovery of cisplatin-induced acute kidney injury. (2017) Korean.J.Intern.Me. 32:1053-1061  FCM ;  Mouse.  

PubMed:28192890

Paraformaldehyde-fixed, paraffin embedded (rat kidney); 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 (Uromucoid) Polyclonal Antibody, Unconjugated (bs-2189R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

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-MCKD2/UMOD Polyclonal Antibody, Unconjugated(bs-2189R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: human kidney tissue;4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-Uromucoid Polyclonal Antibody, Unconjugated(bs-2189R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated(bs-0295G-FITC)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue,C-0033) was used to stain the cell nuclei

Tissue/cell: rat kidney tissue;4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-Uromucoid Polyclonal Antibody, Unconjugated(bs-2189R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated(bs-0295G-Cy3)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue,C-0033) was used to stain the cell nuclei

Blank control: Mouse kidney (blue).
Primary Antibody:Rabbit Anti-Coxsackie Adenovirus Receptor antibody (bs-2189R,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.
Protocol
The cells were fixed with 2% paraformaldehyde for 10 min at 37℃. Primary antibody (bs-2189R, 1μg /1x10^6 cells) were incubated for 30 min at room temperature, followed by 1 X PBS containing 0.5% BSA + 10% goat serum (1 hour) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/FITC antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 40 min at room temperature. Acquisition of 20,000 events was performed.