Anti-CD146 antibody [EPR3208] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

This data was developed using ab75769 the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of formalin fixed paraffin embedded human liver hepatocellular carcinoma labelling CD146 with ab75769 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab75769 Anti-CD146 antibody [EPR3208] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

Immunohistochemistry experiments were used to compare symptomatic carotid plaques (SC) and asymptomatic carotid plaques (AsC)

Asymptomatic lesions presented higher CD146⁺ pericyte infiltration, p

ab75769 used at 1/200 dilution.

(After Figure 2 of Davaine et al)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75769).

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Image from Davaine J.M. et al. PLoS One. 2014 Sep 26;9(9):e107642. doi: 10.1371/journal.pone.0107642. eCollection 2014.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

Intracellular Flow Cytometry analysis of HUVEC cells labelling CD146 with purified ab75769 at 1/50 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75769).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

Intracellular Flow Cytometry analysis of A375 (human malignant melanoma) cells labeling CD146 with unpurified ab75769 at 1/20 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75769).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

This IHC data was generated using the same anti-CD146 antibody clone, EPR3208, in a different buffer formulation (cat# ab75769).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling CD146 with purified ab75769 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

This data was developed using ab75769 the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling CD146 with ab75769 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab75769 Anti-CD146 antibody [EPR3208] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• WB

Lab

Western blot - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75769).

Lanes 1- 2 : Merged signal (red and green). Green - ab75769 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab75769 was shown to react with CD146 in wild-type HeLa cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab261790 (CRISPR/Cas9 edited cell lysate ab256985) lane below 120kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and MCAM CRISPR/Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab75769 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CD146 antibody [EPR3208] (<a href='/en-us/products/primary-antibodies/cd146-antibody-epr3208-ab75769'>ab75769</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MCAM CRISPR/Cas9 edited HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human MCAM (CD146) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-mcam-cd146-knockout-hela-cell-line-ab261790'>ab261790</a>)

Predicted band size: 72 kDa

Observed band size: 120 kDa

false

• WB

Lab

Western blot - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

Lane 1 : Wild-type HAP1 whole cell lysate (40 μg)
Lane 2 : CD146 knockout HAP1 whole cell lysate (40 μg)
Lane 3 : A375 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab75769 observed at 120-72 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab75769 was shown to specifically react with CD146 in wild-type HAP1 cells as signal was lost in CD146 knockout cells. Wild-type and CD146 knockout samples were subjected to SDS-PAGE. ab75769 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75769).

All lanes:

Western blot - Anti-CD146 antibody [EPR3208] - BSA and Azide free (ab210072)

Predicted band size: 72 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal tonsil tissue labelling CD146 with unpurified ab75769.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75769).

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of urinary bladder transitional carcinoma vessels tissue labelling CD146 with unpurified ab75769.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75769).

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of glioma vessels tissue labelling CD146 with unpurified ab75769.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75769).

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of melanoma tissue labelling CD146 with unpurified ab75769 at 1/250. A HRP/AP polymerized secondary antibody was used.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75769).

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal spleen tissue labelling CD146 with unpurified ab75769.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75769).

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD146 antibody [EPR3208] - BSA and Azide free (AB210072)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of breast carcinoma vessels tissue labelling CD146 with unpurified ab75769.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab75769).

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.