Anti-CD8 alpha antibody [EPR21769](AB217344)

Flow cytometric analysis of mouse primary splenocytes labeling CD8 alpha with ab217344 at 1/500 dilution (right panel) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left panel). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Cells were surface stained with CD4-Alexa Fluor® 647, then stained with rabbit IgG (Left) / ab217344 (Right) separately. CD4 and CD8 alpha are mutually exclusive expressed in mouse spleen. Gated on total viable cells.

CD8 alpha was immunoprecipitated from 0.35 mg of mouse thymus lysate with ab217344 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab217344 at 1/2000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: Mouse thymus lysate 10 μg (Input).

Lane 2: ab217344 IP in mouse thymus lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab214344 in mouse thymus lysate.

Exposure time: 5 seconds.

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

The two bands are different isoforms that are consistent with the literature (PMID 3085089).

All lanes:

Immunoprecipitation - Anti-CD8 alpha antibody [EPR21769] (ab217344)

Predicted band size: 26 kDa

Observed band size: 34-38 kDa

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse spleen tissue labeling CD8 alpha with ab217344 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive membrane staining on mouse spleen (PMID: 25616911).

The nuclear counter stain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling CD8 alpha with ab217344 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining on stromal cells of mouse colon. Counter stained with Hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse thymus tissue labeling CD8 alpha with ab217344 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive membrane staining on mouse thymus tissue section (PMID: 25616911).

The nuclear counter stain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

Exposure time : Lane 1: 3 sconds; Lane 2: 40 seconds; Lane 3: 15 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

The two bands are different isoforms that are consistent with the literature (PMID 3085089).

The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.

All lanes:

Western blot - Anti-CD8 alpha antibody [EPR21769] (ab217344) at 1/1000 dilution

Lane 1:

Mouse thymus lysate at 20 µg

Lane 2:

Mouse spleen lysate at 20 µg

Lane 3:

Mouse lymph node lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 26 kDa

Observed band size: 35 kDa