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[IF=0] Garovic, Vesna D., and Muthuvel Jayachandran. "DETECTING PODOCYTE INJURY IN DIABETIC NEPHROPATHY AND GLOMERULONEPHRITIS." U.S. Patent No. 20,170,003,299. 5 Jan. 2017.  Human.  

PubMed:U.S.PatentNo.20,170,003,299.5

Sample: Cerebrum (Rat) Lysate at 40 ug
Primary: Anti-Synaptopodin (bs-3633R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 102 kD
Observed band size: 76 kD

Sample: Brain (Mouse) Lysate at 40 ug
Primary: Anti-Synaptopodin (bs-3633R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 102 kD
Observed band size: 102 kD

Paraformaldehyde-fixed, paraffin embedded (Mouse brain); Antigen retrieval by microwave in sodium citrate buffer (pH6.0) ; Block endogenous peroxidase by 3% hydrogen peroxide for 30 minutes; Blocking buffer (3% BSA) at RT for 30min; Antibody incubation with (Synaptopodin) Polyclonal Antibody, Unconjugated (bs-3633R) at 1:400 overnight at 4°C, followed by conjugation to the secondary antibody (labeled with HRP)and DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Rat brain); 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 (Synaptopodin) Polyclonal Antibody, Unconjugated (bs-3633R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Human 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 (Synaptopodin) Polyclonal Antibody, Unconjugated (bs-3633R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Blank control（black line）:SH-SY5Y.
Primary Antibody (green line): Rabbit Anti-Synaptopodin antibody (bs-3633R)
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.

Positive control: RSC96
Isotype Control Antibody: Rabbit IgG; Secondary Antibody: Goat anti-rabbit IgG-FITC; Dilution: 1:200 in 1 X PBS containing 0.5% BSA
Primary Antibody catalog number: bs-3633R; Dilution: 1μg in 100 μl 1X PBS containing 0.5% BSA

Blank control（black line）:SH-SY5Y.
Primary Antibody (green line): Rabbit Anti-Synaptopodin antibody (bs-3633R)
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.