Recombinant Anti-CD42b antibody [AK2] (ab61402)

Immunocytochemistry analysis of HEL (human Erythroleukemia erythroblast) labelling CD42b with ab61402 at 1/100 (6.3 µg/mL). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat anti Mouse IgG (Alexa Fluor^® 488, ab150113) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. Cells were counterstained with ab179504 Anti-beta IV Tubulin antibody - Microtubule Marker 1/1000 (1 µg/mL), followed by Goat anti-Rabbit, AlexaFluor®594 ab150080 at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
Confocal image showing membranous staining in HEL cells.

Immunohistochemistry image of CD42b staining in a section of frozen normal human spleen performed on a Leica BOND™ system using the standard protocol.

The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab61402, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjµgated 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 cytometry staining of Human peripheral blood mononuclear cell (PBMC) with ab61402 (right) or mouse IgG isotype control (left) at 1/500 dilution, followed by Goat anti mouse IgG (Alexa Fluor® 488, ab150113) at 1/2000 dilution. Cells were stained with mouse IgG (Left) or ab61402 (Right). Then stained with anti-CD41 conjugated to APC.

Gated on viable cells.

Immunocytochemistry analysis of Mouse splenocytes labelling CD42b with ab61402 at 1/100 (6.3 µg/mL). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat anti Mouse IgG (Alexa Fluor^® 488, ab150113) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. Cells were counterstained with ab179504 Anti-beta IV Tubulin antibody - Microtubule Marker 1/1000 (1 µg/mL), followed by Goat anti-Rabbit, AlexaFluor®594 ab150080 at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
Confocal image showing membranous staining in mouse splenocytes.

Flow cytometry staining of human whole blood with ab61402 (right) or mouse IgG1 kappa; (ab170190) isotype (left). Red blood cells of 200 µl blood were lysed, then cells were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 µll normal goat serum to block Fc receptors and non-specific protein-protein interaction followed by the antibody (ab61402) or mouse IgG1 kappa; (ab170190) isotype (1x10⁶ in 100 µl; at 1 µg/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.

The cells were simultaneously stained with CD41/CD61  APC. Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on granulocytes.

Negative control image. Immunohistochemistry image of CD42b staining in a section of frozen normal human cerebral cortex performed on a Leica BOND™ system using the standard protocol.

The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab61402, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjµgated 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.