VIM | Vimentin Mouse mAb(bsm-3

Blank control:Hela.
_x000D_ Primary Antibody (green line): Mouse Anti-Vimentin antibody (bsm-33170M)
_x000D_ Dilution: 1ug/Test;
_x000D_ Secondary Antibody : Goat anti-Mouse IgG-FITC
_x000D_ Dilution: 0.5ug/Test.
_x000D_ Protocol
_x000D_ 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._x000D_

Sample:
_x000D_ Hela(Human) Cell Lysate at 30 ug
_x000D_ Hela KO Vimentin (Human) Cell Lysate at 30 ug
_x000D_ Primary: Anti- Vimentin (bsm-33170M) at 1/1000 dilution
_x000D_ Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution
_x000D_ Predicted band size: 53 kD
_x000D_ Observed band size: 58 kD
_x000D_

Hela 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 (Vimentin) monoclonal Antibody, Unconjugated (bsm-33170M) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Mouse IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

P‌‌araformaldehyde-fixed, paraffin embedded Human Cervical Cancer; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Vimentin Monoclonal Antibody, Unconjugated(bsm-33170M) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Mouse, sp-0024) and DAB (C-0010) staining.

P‌‌araformaldehyde-fixed, paraffin embedded Human Tonsil; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Vimentin Monoclonal Antibody, Unconjugated(bsm-33170M) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Mouse, sp-0024) and DAB (C-0010) staining.

P‌‌araformaldehyde-fixed, paraffin embedded Human Colon; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Vimentin Monoclonal Antibody, Unconjugated(bsm-33170M) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Mouse, sp-0024) and DAB (C-0010) staining.

P‌‌araformaldehyde-fixed, paraffin embedded Human Kidney; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Vimentin Monoclonal Antibody, Unconjugated(bsm-33170M) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Mouse, sp-0024) and DAB (C-0010) staining.

P‌‌araformaldehyde-fixed, paraffin embedded Human Uterus; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Vimentin Monoclonal Antibody, Unconjugated(bsm-33170M) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Mouse, sp-0024) and DAB (C-0010) staining.

P‌‌araformaldehyde-fixed, paraffin embedded Uterine body cancer; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Vimentin Monoclonal Antibody, Unconjugated(bsm-33170M) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Mouse, sp-0024) and DAB (C-0010) staining.

4% P‌‌araformaldehyde-fixed Hela (left) cell and Vimentin knockout Hela(right) cell; Triton X-100 at r.t. for 20 min; Antibody incubation with (Vimentin) monoclonal Antibody, unconjugated (bsm-33170M) 1:100, 90 min at 37°C; followed by conjugated Goat Anti-Mouse IgG antibody (green, bs-60296G-FITC) at 37°C for 90 min, DAPI (blue, C02-04002) was used to stain the cell nuclei.

25 ug total protein per lane of various lysates (see on figure) probed with Vimentin monoclonal antibody, unconjugated (bsm-33170M) at 1:1000 dilution and 4°C overnight incubation. Followed by conjugated secondary antibody incubation at r.t. for 60 min.

P‌‌araformaldehyde-fixed, paraffin embedded Human Colon; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Vimentin Monoclonal Antibody, Unconjugated (bsm-33170M) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Mouse IgG antibody (green, bs-0296G-BF488), DAPI (blue, C02-04002) was used to stain the cell nuclei.

P‌‌araformaldehyde-fixed, paraffin embedded Human Colon Cancer; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Vimentin Monoclonal Antibody, Unconjugated (bsm-33170M) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Mouse IgG antibody (green, bs-0296G-BF488), DAPI (blue, C02-04002) was used to stain the cell nuclei.

P‌‌araformaldehyde-fixed, paraffin embedded Human Colon Cancer; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; The section was incubated with Vimentin Monoclonal Antibody, Unconjugated (bsm-33170M) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Mouse IgG antibody (rose red, bs-0296G-BF647), DAPI (blue, C02-04002) was used to stain the cell nuclei.

P‌‌araformaldehyde-fixed, paraffin embedded Human Small Intestine; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; The section was incubated with Vimentin Monoclonal Antibody, Unconjugated (bsm-33170M) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Mouse IgG antibody (rose red, bs-0296G-BF647), DAPI (blue, C02-04002) was used to stain the cell nuclei.