Anti-ICAM1 antibody [EPR4776]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ICAM1 antibody [EPR4776] (AB109361)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling ICAM1 with Purified ab109361 at 1/100 dilution (6.79 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ICAM1 antibody [EPR4776] (AB109361)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue sections labeling ICAM1 with Purified ab109361 at 1/100 dilution (6.79 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ICAM1 antibody [EPR4776] (AB109361)

Intracellular Flow Cytometry analysis of Ramos (Human Burkitt's lymphoma B lymphocyte) cells labeling ICAM1 with Purified ab109361 at 1/70 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ICAM1 antibody [EPR4776] (AB109361)

Immunocytochemistry analysis of Raji (Human Burkitt's lymphoma B lymphocyte) cells labeling ICAM1 with Purified ab109361 at 1/100 dilution (6.79 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 dilution (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/mL). DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IP

Lab

Immunoprecipitation - Anti-ICAM1 antibody [EPR4776] (AB109361)

Purified ab109361 at 1/30 dilution (2µg) immunoprecipitating ICAM1 in Raji whole cell lysate.
Lane 1 (input) : Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysate 10µg
Lane 2 (+) : ab109361 + Raji whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109361 in Raji whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 89 kDa

All lanes:

Immunoprecipitation - Anti-ICAM1 antibody [EPR4776] (ab109361)

Predicted band size: 57 kDa

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• WB

Lab

Western blot - Anti-ICAM1 antibody [EPR4776] (AB109361)

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

All lanes:

Western blot - Anti-ICAM1 antibody [EPR4776] (ab109361) at 1/1000 dilution

Lane 1:

HUVEC (Human umbilical vein endothelial cell) whole cell lysate at 15 µg

Lane 2:

HUVEC (Human umbilical vein endothelial cell) treated with 10ng/ml TNF-α for 18 hours whole cell lysate at 15 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

false

• WB

Lab

Western blot - Anti-ICAM1 antibody [EPR4776] (AB109361)

False colour image of Western blot : Anti-ICAM1 antibody [EPR4776] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109361 was shown to bind specifically to ICAM1. A band was observed at 90 kDa in wild-type HeLa cell lysates with no signal observed at this size in Icam1 knockout cell line ab261742 (knockout cell lysate ab256947). The band observed in the knockout lysate lane below 90 kDa is likely to represent a truncated form of ICAM1. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and Icam1 knockout HeLa cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-ICAM1 antibody [EPR4776] (ab109361) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ICAM1 knockout HeLa cell lysate at 20 µg

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

Ramos cell lysate at 20 µg

Predicted band size: 57 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Anti-ICAM1 antibody [EPR4776] (AB109361)

False colour image of Western blot : Anti-ICAM1 antibody [EPR4776] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109361 was shown to bind specifically to ICAM1. A band was observed at 90 kDa in wild-type HeLa cell lysates with no signal observed at this size in Icam1 CRISPR-Cas9 edited cell line ab261742 (CRISPR-Cas9 edited cell lysate ab256947). The band observed in the CRISPR-Cas9 edited lysate lane below 90 kDa is likely to represent a truncated form of ICAM1. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and Icam1 CRISPR-Cas9 edited HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-ICAM1 antibody [EPR4776] (ab109361) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ICAM1 CRISPR-Cas9 edited HeLa 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:

A549 cell lysate at 20 µg

Lane 4:

Ramos cell lysate at 20 µg

Predicted band size: 57 kDa

Observed band size: 90 kDa

false

• WB

Unknown

Western blot - Anti-ICAM1 antibody [EPR4776] (AB109361)

All lanes:

Western blot - Anti-ICAM1 antibody [EPR4776] (ab109361) at 1/1000 dilution

All lanes:

Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate at 15 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

false