Recombinant Anti-CD8 alpha antibody [EPR21769] - BSA and Azide free (ab230156)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217344).

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.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217344).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217344).

Clone EPR21769 (ab230156) has been successfully conjugated by Abcam. This image was generated using Anti-CD8 alpha antibody [EPR21769] (Alexa Fluor® 647). Please refer to ab237365 for protocol details.

IHC image of CD8 alpha staining in a section of formalin-fixed paraffin-embedded normal mouse spleen.

The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer (pH9, epitope retrieval solution 2) for 20mins, performed on a Leica BOND^™. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab237365 at 1/100 dilution (shown in red). Nuclear DNA was labeled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

Clone EPR21769 (ab230156) has been successfully conjugated by Abcam. This image was generated using Anti-CD8 alpha antibody [EPR21769] (Alexa Fluor® 488). Please refer to ab237364 for protocol details.

IHC image of CD8 alpha staining in a section of formalin-fixed paraffin-embedded normal mouse spleen.

The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer (pH9, epitope retrieval solution 2) for 20mins, performed on a Leica BOND^™. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab237364 at 1/100 dilution (shown in green). Nuclear DNA was labeled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217344).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217344).

Immunohistochemical analysis of paraffin-embedded mouse spleen 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 the white pulp of mouse spleen (PMID: 23482450; PMID: 25826597). Counter stained with Hematoxylin.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab217344).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.