重组Anti-HLA-DR抗体[TAL 1B5] - BSA and Azide free (ab176408)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab20181).

ab20181 staining HLA DR in Raji cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised in 0.1% Triton X-100 for 5 minutes and then blocked in 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 ab20181 at 1/1000 dilution and ab7291, Mouse monoclonal to alpha Tubulin at 1/1000 dilution. Cells were then incubated with ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed, at 1/1000 dilution (shown in red) and ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488), preadsorbed at 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Lanes 1-3: Merged signal (red and green). Green - ab20181 observed at 35 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37 kDa.

ab20181 was shown to react with HLA-DR in Western blot. Membranes were blocked with 3% milk in TBS-T (0.1% Tween^®) before incubation with ab20181 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at 1 µg/ml and a 1:20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1:20000 dilution for 1 h at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human skin tissue labeling HLA-DR with ab20181 at 0.1 µg/ml followed by Leica DS9800 (Bond™ Polymer Refine Detection). 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 ab20181, 0.1ug/ml, for 15 mins at room temperature and was then 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.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling HLA-DR with ab20181 at 0.1 µg/ml followed by Leica DS9800 (Bond™ Polymer Refine Detection). 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 ab20181, 0.1ug/ml, for 15 mins at room temperature and was then 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.

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab20181 (right) or mouse IgG1κ (ab170190) isotype (left). PBMCs were incubated for 30 min on ice in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by followed by staining with CD19 APC. PBMCs were then fixed in 4.2% formaldehyde and permeabilised in 0.1% saponin before staining with the antibody (ab20181) or mouse IgG1κ (ab170190) isotype (1x10⁶ in 100μl; at 0.008μg/ml) for 30 min on ice. The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor ® 488, pre-adsorbed) (ab150117) was used at 1:2000 dilution for 30 min on ice. Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on alive lymphocytes.