Recombinant Anti-CD19 antibody [EPR23174-145] (ab245235)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized perfusion fixed frozen Mouse spleen tissue labeling CD19 with ab245235 at 1/250 (1.82 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). Positive staining on mouse spleen. is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Blocking and dilution buffer: 5% NFDM/TBS.

Exposure time: 48 seconds.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 26516065).

Negative control: CTLL-2 (PMID: 1370948) is treated as a negative cell line according to the literature, that CD19 is a B lymphocyte cell-surface marker that is expressed early during pre-B-cell differentiation, not in T cells.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling CD19 with ab245235 at 1/1000 dilution (0.446 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). Positive staining on mouse spleen (PMID: 18648532). The section was incubated with ab245235 for 10 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized mouse PBMC (mouse primary peripheral blood mononuclear cell) cells labeling CD19 with ab245235 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in some subsets of mouse PBMCs. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Flow cytometric analysis of mouse peripheral blood mononuclear cell (PBMC) cells labeling CD19 with ab245235 at 1/500 dilution (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Cells were stained with rabbit IgG (Left) or ab245235 (Right). Then stained with anti-CD3 conjugated to Alexa Fluor^® 647. CD19 and CD3 are mutually exclusive expressed in mouse PBMC.

Gated on viable cells.

CD19 was immunoprecipitated from 0.35 mg mouse spleen lysate with ab245235 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab245235 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Mouse spleen lysate 10µg

Lane 2: ab245235 IP in mouse spleen lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab245235 in mouse spleen lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 48 seconds.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 26516065).