Anti-CD9 antibody [EPR23105-121]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD9 antibody [EPR23105-121] (AB236630)

Immunohistochemical analysis of paraffin-embedded Human bladder carcinoma tissue labeling CD9 with ab236630 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human bladder carcinoma. The section was incubated with ab236630 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD9 antibody [EPR23105-121] (AB236630)

Immunohistochemical analysis of formalin fixed paraffin embedded human bladder carcinoma labelling CD9 with ab236630 at a concentration of 0.5 µg/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, pH 8.5 for 32mins.

ab236630 anti-CD9 [EPR23105-121] 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).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD9 antibody [EPR23105-121] (AB236630)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (human colorectal carcinoma epithelial cell) cells labelling CD9 with ab236630 at 1/500 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in HCT 116 cell line. Negative control : Raji (PMID : 8921952). ab195889 Anti-alpha Tubulin antibody [DM1A] - 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 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

• Flow Cyt

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Flow Cytometry - Anti-CD9 antibody [EPR23105-121] (AB236630)

Flow cytometric analysis of Raji (human Burkitt's lymphoma B lymphocyte, Left) / HCT 116 (human colorectal carcinoma epithelial cell, Right) cells labelling CD9 with ab236630 at 1/50 dilution 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 was used as the secondary antibody.

Negative control : Raji (PMID : 8921952).

Gated on viable cells.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD9 antibody [EPR23105-121] (AB236630)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling CD9 with ab236630 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on platelets of human spleen. The section was incubated with ab236630 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

• Flow Cyt

Lab

Flow Cytometry - Anti-CD9 antibody [EPR23105-121] (AB236630)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab236630 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left).

PBMCs were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab236630 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 0.2 μg/ml (1/10,500)) for 30min on ice.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/2000 for 30min on ice.

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• IP

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Immunoprecipitation - Anti-CD9 antibody [EPR23105-121] (AB236630)

CD9 was immunoprecipitated from 0.35 mg HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate with ab236630 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab236630 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1 : HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate 10ug.

Lane 2 : ab236630 IP in HCT 116 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab236630 in HCT 116 whole cell lysate.

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

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-CD9 antibody [EPR23105-121] (ab236630)

Predicted band size: 25 kDa

Observed band size: 22 kDa

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• WB

Lab

Western blot - Anti-CD9 antibody [EPR23105-121] (AB236630)

Lanes 1 - 4 : Merged signal (red and green). Green - ab236630 observed at 18 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab236630 was shown to react with CD9 in wild-type HeLa cells in Western blot with loss of signal observed in CD9 knockout cell line ab255375 (CD9 knockout cell lysate ab263754). Wild-type HeLa and CD9 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab236630 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-CD9 antibody [EPR23105-121] (ab236630) at 1/1000 dilution

Lane 1:

Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

CD9 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CD9 knockout HeLa cell line (<a href='/ko/products/cell-lines/human-cd9-knockout-hela-cell-line-ab255375'>ab255375</a>)

Lane 3:

A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 25 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Anti-CD9 antibody [EPR23105-121] (AB236630)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 3275469). Exposure time : 3 minutes.

All lanes:

Western blot - Anti-CD9 antibody [EPR23105-121] (ab236630) at 1/1000 dilution

Lane 1:

Human tonsil lysate at 20 µg

Lane 2:

Human colon lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 25 kDa

Observed band size: 22 kDa

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• WB

Lab

Western blot - Anti-CD9 antibody [EPR23105-121] (AB236630)

Blocking and diluting buffer and concentration : 5% NFDM/TBST This blot was developed using a higher sensitivity ECL substrate. The molecular weight observed is consistent with what has been described in the literature (PMID : 3275469).

Negative control : Raji (PMID : 8921952). Exposure time : 3 minutes

All lanes:

Western blot - Anti-CD9 antibody [EPR23105-121] (ab236630) at 1/1000 dilution

Lane 1:

HCT 116 (human colorectal carcinoma epithelial cell) 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='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 25 kDa

Observed band size: 22 kDa

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