Anti-CD8 alpha antibody [SP16] - BSA and Azide free

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-CD8 alpha antibody [SP16] - BSA and Azide free (AB307844)

This data was developed using ab101500, the same antibody clone in a different buffer formulation.

10-color fluorescence multiplex immunohistochemical analysis of human lung cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of anti-FOXP3 (ab215206; Cyan; TG540N), anti-PD1 (ab52587; Red; TG700N), anti-CD163 (ab182422; Brown; TG650N), anti-HLA-DR (ab92511; Yellow; TG570N), anti-CD4 (ab133616; Violet; TG620N), anti-CD8 alpha (ab101500; Purple; TG540S), anti-CD20 (ab9475; Grey; TG660S), anti-CD68 (ab192847; Green; TG520N), anti-Cytokeratin 19 (ab52625; Light blue; TG440N). TG470SN (dark blue) was used as a nuclear counter stain. The inset image shows the separate CD68 signal.

The section was incubated in nine rounds of staining; in the order of ab215206 (1/100 dilution), ab52587 (1/200 dilution), ab182422 (1/300 dilution), ab92511 (1/200 dilution), ab133616 (1/600 dilution), ab101500 (1/300 dilution), ab9475 (1/100 dilution), ab192847 (1/300 dilution), ab52625 (1/400 dilution); each using a separate fluorescent tyramide signal amplification system.

Sodium citrate antigen retrieval (pH6.0) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

Image acquisition was performed with TissueFAXS Spectra (TissueGnostics).

This image is courtesy of TissueGnostics Asia Pacific Limited

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [SP16] - BSA and Azide free (AB307844)

This data was developed using ab101500, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human tonsil labeling CD8 with ab101500 at 1/100 (21 μg/ml). The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab101500 for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. DAB was used as the chromogen. Counterstained with Hematoxylin and mounted with DPX.

• Flow Cyt

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Flow Cytometry - Anti-CD8 alpha antibody [SP16] - BSA and Azide free (AB307844)

This data was developed using ab101500, the same antibody clone in a different buffer formulation.

This image was generated using a previous batch manufactured using hybridoma production method.

Human peripheral blood lymphocytes staining CD8 alpha with ab101500 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID : 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lysed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (ab101500, 1/1000 dilution) for 30 min at 4°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD8 alpha antibody [SP16] - BSA and Azide free (AB307844)

This data was developed using ab101500, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling CD8 alpha with ab101500 at a dilution of 1/100. 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, pH8.5 for 32mins.

ab101500 Anti-CD8 alpha antibody [SP16] 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)