Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labelling ICAM1 with ab282575 at 1/600 (0.902 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on normal human glomerulus. The section was incubated with ab282575 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of Raji (Human Burkitt's lymphoma B lymphocyte) cells labelling ICAM1 with ab282575 at 1/500 dilution (0.1 μg)/(Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labelling ICAM1 with ab282575 at 1/600 (0.902 μg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human breast cancer (PMID : 30082828). The section was incubated with ab282575 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji cells labelling ICAM1 with ab282575 at 1/500 (1.082 μg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in Raji cells.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• ICC

Lab

Immunocytochemistry - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized ICAM1 KO HeLa cells labelling ICAM1 with ab282575 at 1/500 (1.082 μg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in wild-type HeLa cells, and no staining in ICAM1 knockout HeLa cells is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

ICAM1 was immunoprecipitated from 0.35 mg Raji (human burkitt's lymphoma b lymphocyte) whole cell lysate 10 μg with ab282575 at 1/30 dilution (2 μg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab282575 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Raji whole cell lysate 10 μg

Lane 2 : ab282575 IP in Raji whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab282575 in Raji whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-ICAM1 antibody [EPR24639-3] (<a href='/en-us/products/primary-antibodies/icam1-antibody-epr24639-3-ab282575'>ab282575</a>)

Predicted band size: 57 kDa

Observed band size: 90 kDa

false

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of C6 (Rat glial tumor glial cell) cells labelling ICAM1 with ab282575 at 1/500 dilution (0.1 μg)/(red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of rat splenocytes cells labelling ICAM1 with ab282575 at 1/500 dilution (0.1 μg)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. A goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

• WB

Lab

Western blot - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

Lanes 1-3 : Merged signal (red and green). Green - ab282575 observed at 90kDa. Red - loading control ab8245 observed at 36 kDa.

ab282575 Anti-ICAM1 antibody [EPR24639-3] was shown to react with ICAM1 in wild-type Hela cells in Western blot. Loss of signal was observed when knockout cell line ab261742 (knockout cell lysate ab256947) was used. Wild-type and ICAM1 knockout samples were subjected to SDS-PAGE.

ab282575 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1 in 1000 dilution and 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 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ICAM1 antibody [EPR24639-3] (<a href='/en-us/products/primary-antibodies/icam1-antibody-epr24639-3-ab282575'>ab282575</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

ICAM1 knockout HeLa whole cell lysate at 20 µg

Lane 2:

Western blot - Human ICAM1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-icam1-knockout-hela-cell-line-ab261742'>ab261742</a>)

Lane 3:

Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 57 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

ab282575 Anti-ICAM1 antibody [EPR24639-3] was shown to react with ICAM1 in wild-type Hela cells in Western blot. Loss of signal was observed when knockout cell line ab261742 (knockout cell lysate ab256947) was used. Wild-type and ICAM1 knockout samples were subjected to SDS-PAGE.

Exposure time : 15 seconds.

All lanes:

Western blot - Anti-ICAM1 antibody [EPR24639-3] (<a href='/en-us/products/primary-antibodies/icam1-antibody-epr24639-3-ab282575'>ab282575</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

ICAM1 knockout Hela whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 57 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Exposure time : 26 seconds

All lanes:

Western blot - Anti-ICAM1 antibody [EPR24639-3] (<a href='/en-us/products/primary-antibodies/icam1-antibody-epr24639-3-ab282575'>ab282575</a>) at 1/1000 dilution

All lanes:

human kidney tissue lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 57 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (AB282599)

This data was developed using ab282575, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Exposure time : 15 seconds

All lanes:

Western blot - Anti-ICAM1 antibody [EPR24639-3] (<a href='/en-us/products/primary-antibodies/icam1-antibody-epr24639-3-ab282575'>ab282575</a>) at 1/1000 dilution

Lane 1:

Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 57 kDa

Observed band size: 90 kDa

false