All lanes : Anti-CD3 antibody [SP7] (ab16669) at 1/25 dilution

Lane 1 : THP1 whole cell lysate (-ve control)
Lane 2 : Raji whole cell lysate (-ve control)
Lane 3 : Jurkat whole cell lysate
Lane 4 : Human Thymus tissue lysate
Lane 5 : Mouse Thymus tissue lysate
Lane 6 : Rat Thymus tissue lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 19 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?

Lanes 1 - 6: Merged signal (red and green). Green – ab16669 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab16669 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.

This data was developed using the undiluted version of this antibody (ab16669).

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human tonsil tissue, staining CD3 with ab21703.

Human peripheral blood lymphocytes stained with ab16669 (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 lyzed 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 (ab16669, 1/1000 dilution) for 30 min at 4°C. The secondary antibody used was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (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.

This data was developed using the undiluted version of this antibody (ab16669).

This data was developed using the undiluted version of this antibody (ab16669).

Fluorescence multiplex immunohistochemical analysis of normal human tonsil tissue (formalin-fixed paraffin-embedded section).

Merged staining of anti-PD1 (ab237728; orange; Opal™520), anti-PDL1 (ab237726; green; Opal™540), anti-CD68 (ab192847; yellow; Opal™570), anti-CD3 (ab16669; red; Opal™620), anti-Ki67 (ab16667; light blue; Opal™650) and anti-PanCK (ab7753; grey; Opal™690).

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 7-color automation IHC kit (NEL821001KT, Akoya Biosciences®).

The section was incubated in six rounds of staining; in the order of ab237728 (1/500 dilution), ab237726 (1/500 dilution), ab192847 (1/300 dilution), ab16669 (1/300 dilution), ab16667 (1/200 dilution) and ab7753 (1/200 dilution); each using a separate fluorescent tyramide signal amplification system.

Sodium citrate antigen retrieval (Leica ER1, pH6.0, 30 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

DAPI (dark blue) was used as a nuclear counter stain.

Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra Polaris.

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human thymus tissue, staining CD3 with ab21703.

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human Hodgkin's lymphoma tissue, staining CD3 with ab21703.

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human uterus tissue, staining CD3 with ab21703.