Anti-CD8 alpha antibody [RM1129]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [RM1129] (AB316778)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CD8 alpha with ab316778 at 1/100 (4.95 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil.
The section was incubated with ab316778 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CD8 alpha antibody [RM1129] (AB316778)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Human PBMC (human peripheral blood mononuclear cell) cells labelling CD8 alpha with ab316778 at 1/500 dilution (0.1ug)/ Right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Cells are co-stianed with CD4 conjugated to Alexa Fluor®647.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [RM1129] (AB316778)

Immunohistochemical analysis of paraffin-embedded Human gastric cancer tissue labeling CD8 alpha with ab316778 at 1/100 (4.95 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on T lymphocytes of human gastric cancer.
The section was incubated with ab316778 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [RM1129] (AB316778)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling CD8 alpha with ab316778 at 1/100 (4.95 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on T lymphocytes of human colon.
The section was incubated with ab316778 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD8 alpha antibody [RM1129] (AB316778)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Human PBMC (human primary peripheral blood mononuclear cell) cells labellingCD8 alpha with ab316778 at 1/100 (4.95 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution.

Confocal image showing membranous and cytoplasmic staining in subsets of human PBMCs (shown in green). The counterstain was observed in red. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution. The Nuclear counterstain was DAPI.

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-CD8 alpha antibody [RM1129] (AB316778)

CD8 alpha was immunoprecipitated from 0.35 mg Human thymus tissue lysate with ab316778 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316778 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Human thymus tissue lysate
Lane 2 : ab316778 IP in Human thymus tissue lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab316778 in human thymus tissue lysate

All lanes:

Immunoprecipitation - Anti-CD8 alpha antibody [RM1129] (ab316778) at 1/30 dilution

All lanes:

Human thymus tissue lysate with 5% NFDM/TBST

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 93s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [RM1129] (AB316778)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling CD8 alpha with ab316778 at 1/100 (4.95 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen.
The section was incubated with ab316778 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD8 alpha antibody [RM1129] (AB316778)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse splenocyte cells labellingCD8 alpha with ab316778 at 1/100 (4.95 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution.

Confocal image showing membranous and cytoplasmic staining in subsets of mouse splenocytes (shown in green). The counterstain was observed in red. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution. The Nuclear counterstain was DAPI.

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD8 alpha antibody [RM1129] (AB316778)

Flow cytometric analysis of Mouse splenocytes cells labelling CD8 alpha with ab316778 at 1/50 dilution (1 ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells. Cells are co-stained with CD4 conjugated to Alexa Fluor®647.

• IP

Supplier Data

Immunoprecipitation - Anti-CD8 alpha antibody [RM1129] (AB316778)

CD8 alpha was immunoprecipitated from 0.35 mg Mouse spleen tissue lysate with ab316778 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316778 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse spleen tissue lysate
Lane 2 : ab316778 IP in Mouse spleen tissue lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab316778 in mouse spleen tissue lysate

All lanes:

Immunoprecipitation - Anti-CD8 alpha antibody [RM1129] (ab316778) at 1/30 dilution

All lanes:

Mouse spleen tissue lysate with 5% NFDM/TBST

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-CD8 alpha antibody [RM1129] (AB316778)

Negative control : brain, heart and kidney.

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lane 1-4 : 180 seconds Lane 5 : 10 seconds

All lanes:

Western blot - Anti-CD8 alpha antibody [RM1129] (ab316778) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg with 5% NFDM/TBST

Lane 2:

Mouse heart tissue lysate at 20 µg with 5% NFDM/TBST

Lane 3:

Mouse kidney tissue lysate at 20 µg with 5% NFDM/TBST

Lane 4:

Mouse spleen tissue lysate at 20 µg with 5% NFDM/TBST

Lane 5:

Mouse thymus tissue lysate at 20 µg with 5% NFDM/TBST

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 1/100000 dilution

Observed band size: 28-32 kDa,36 kDa

false