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Sample: BV-2(Rat) Cell Lysate at 30 ug Primary: Anti-TUBB3 (Neuronal Marker) (bs-4512R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 50-55 kD Observed band size: 50 kD

Sample: U251(Human) Cell Lysate at 30 ug Primary: Anti-TUBB3 (Neuronal Marker) (bs-4512R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 50-55 kD Observed band size: 50 kD

Tissue/cell: rat brain 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-TUBB3/beta III Tubulin(Neuronal Marker) Polyclonal Antibody, Unconjugated(bs-4512R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) 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 (TUBB3 (Neuronal Marker)) Polyclonal Antibody, Unconjugated (bs-4512R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Tissue/cell: BV-2 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (TUBB3) Polyclonal Antibody, Unconjugated (bs-4512R) 1:200, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody (bs-0295G-FITC) at 37°C for 90 minutes, DAPI (5ug/ml, blue, C-0033) was used to stain the cell nuclei.

SH-SY5Y cell; 4% Paraformaldehyde-fixed; Ice-cold methanol at -20℃ for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (TUBB3) polyclonal Antibody, Unconjugated (bs-4512R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Paraformaldehyde-fixed, paraffin embedded (Mouse 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 (TUBB3) Polyclonal Antibody, Unconjugated (bs-4512R) at 1:400 overnight at 4°C, followed by a conjugated Goat Anti-Rabbit IgG antibody (bs-0295G-AF594) for 90 minutes, and DAPI for nuclei 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 (TUBB3) Polyclonal Antibody, Unconjugated (bs-4512R) at 1:400 overnight at 4°C, followed by a conjugated Goat Anti-Rabbit IgG antibody (bs-0295G-AF594) for 90 minutes, and DAPI for nuclei staining.

Blank control:SH-SY5Y. Primary Antibody (green line): Rabbit Anti-TUBB3 (Neuronal Marker) antibody (bs-4512R) Dilution: 1ug/Test; Secondary Antibody : Goat anti-rabbit IgG-FITC Dilution: 0.5ug/Test. 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.

Blank control(blue): U-87MG Cells(fixed with 2% paraformaldehyde (10 min)). P rimary Antibody: Rabbit Anti-MGLUR3/AF647 Conjugated antibody (bs-4512R/AF647), Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit IgG/AF647(orange) ,used under the same conditions.

Blank control:SH-SY5Y. Primary Antibody (green line): Rabbit Anti-TUBB3 (Neuronal Marker) antibody (bs-4512R) Dilution: 1ug/Test; Secondary Antibody : Goat anti-rabbit IgG-FITC Dilution: 0.5ug/Test. 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.