Sample: Lane 1: Mouse Cerebrum tissue lysates Lane 2: Rat Cerebrum tissue lysates Lane 3: Rat Cerebellum tissue lysates Lane 4: Human U251 cell lysates Primary: Anti-NF-L (bs-41217R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 68 kDa Observed band size: 62 kDa

Paraformaldehyde-fixed, paraffin embedded (human cerebellum); 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 (NF-L) Polyclonal Antibody, Unconjugated (bs-41217R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat cerebellum); 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 (NF-L) Polyclonal Antibody, Unconjugated (bs-41217R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand 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 (NF-L) Polyclonal Antibody, Unconjugated (bs-41217R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum); 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 (NF-L) Polyclonal Antibody, Unconjugated (bs-41217R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat spinal cord); 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 (NF-L) Polyclonal Antibody, Unconjugated (bs-41217R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse spinal cord); 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 (NF-L) Polyclonal Antibody, Unconjugated (bs-41217R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded Mouse Cerebellum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with NF-L Polyclonal Antibody, Unconjugated (bs-41217R) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Rabbit IgG antibody (Purple, bs-0295D-Cy5), DAPI (blue, C02-04002) was used to stain the cell nuclei.

Paraformaldehyde-fixed, paraffin embedded Mouse Cerebrum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with NF-L Polyclonal Antibody, Unconjugated (bs-41217R) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Rabbit IgG antibody (Purple, bs-0295D-Cy5), DAPI (blue, C02-04002) was used to stain the cell nuclei.

Paraformaldehyde-fixed, paraffin embedded Human Left Parietal Lobe; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with NF-L Polyclonal Antibody, Unconjugated (bs-41217R) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Rabbit IgG antibody (Purple, bs-0295D-Cy5), DAPI (blue, C02-04002) was used to stain the cell nuclei.

Paraformaldehyde-fixed, paraffin embedded Rat Cerebellum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with NF-L Polyclonal Antibody, Unconjugated (bs-41217R) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Rabbit IgG antibody (Purple, bs-0295D-Cy5), DAPI (blue, C02-04002) was used to stain the cell nuclei.

Paraformaldehyde-fixed, paraffin embedded Rat Cerebrum; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with NF-L Polyclonal Antibody, Unconjugated (bs-41217R) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Rabbit IgG antibody (Purple, bs-0295D-Cy5), DAPI (blue, C02-04002) was used to stain the cell nuclei.

Blank control（black line）:U251. Primary Antibody (green line): Rabbit Anti-NF-L antibody (bs-41217R) Dilution:1ug/Test; Secondary Antibody（white blue line）: Goat anti-rabbit IgG-FITC 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.