Anti-CD8 alpha antibody [OX-8]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [OX-8] (AB33786)

IHC image of CD8 alpha staining in a section of formalin-fixed paraffin-embedded normal Rat Spleen performed on a Leica BOND™ system using the standard Protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab33923, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody. For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• Flow Cyt

Lab

Flow Cytometry - Anti-CD8 alpha antibody [OX-8] (AB33786)

Flow cytometry staining of Lewis rat splenocytes with ab33786 (right) or mouse IgG1 kappa; (ab170190) isotype (left). Cells were incubated for 30 min on ice in 1x PBS containing 10 % rat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab33786) or mouse IgG1 kappa; (ab170190) isotype (1x 10⁶ in 100 μl at 0.008 μg/ml) for 30 min on ice.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150177) was used at 1/2000 dilution for 30 min on ice.

The cells were simultaneously stained with CD4.

Acquisition of >30,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. Events were gated on live CD3 positive T cells.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD8 alpha antibody [OX-8] (AB33786)

ab33786 staining CD8 alpha in Rat splenocytes cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab33786 at 5μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 100% methanol (5 min).

• WB

Lab

Western blot - Anti-CD8 alpha antibody [OX-8] (AB33786)

This blot was produced using a 4-12% Bis-tris under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was blocked for an hour using 3% milk before ab33786 and ab176560 (Rabbit anti-alpha Tubulin loading control) were incubated overnight at 4°C at a 1 μg/ml concentration and 1/20000 dilution respectively. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CD8 alpha antibody [OX-8] (ab33786) at 1 µg/mL

Lane 1:

Rat thymus tissue lysate at 20 µg

Lane 2:

Rat brain tissue lysate at 20 µg

Predicted band size: 26 kDa

Observed band size: 32 kDa

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• IHC-P

AbReview26014****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [OX-8] (AB33786)

ab33786 staining CD8 alpha in rat spleen tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

Tissue was fixed with 10% neutral buffered formalin and a heat mediated antigen retrieval step was performed using citrate buffer. Samples were then permeabilized with Triton X-100, blocked using 5% serum for 30 minutes at 25°C and then incubated with ab33786 at a 1/100 dilution for 1 hour at 25°C. The secondary used was a biotin-conjugated rabbit anti-mouse polyclonal, used at a 1/300 dilution.

Image courtesy of an anonymous Abreview.