Recombinant Anti-CD63 antibody [EPR22458-280] (ab252919)

Flow cytometric analysis of SK-MEL-28 (human malignant melanoma cell line) cells labeling CD63 with ab252919 at 1/60 (red) compared with a Rabbit monoclonal IgG Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

Left panel: No fixation/permeabilization; Gated on viable cells for surface CD63 staining.

Right panel: 4% paraformadehyde fixation / 90% methanol permeabilization.

Flow cytometric analysis of human peripheral blood mononuclear cells labeling CD63 with ab252919 at 1/60 (right) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left). Goat Anti-Rabbit IgG Fc (Alexa Fluor^® 488) preadsorbed (ab150097), at 1/5000 dilution was used as the secondary antibody.

Cells were stained with rabbit IgG (Left) or ab252919 (Right). Then stained with anti-CD14 conjugated to Alexa Fluor^® 647.

Gated on viable cells.

Immunohistochemical analysis of paraffin-embedded human melanoma tissue labeling CD63 with ab252919 at 1/2000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human melanoma (PMID: 22425483) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

The section was incubated with ab252919 for 30 minutes at RT.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling CD63 with ab252919 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Negative control: No staining on normal epithelial cells and stromal cells in human breast (PMID: 22957045).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SK-MEL-28 (human malignant melanoma cell line) cells labeling CD63 with ab252919 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and membranous staining in SK-MEL-28 cell line. The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SK-MEL-28 (human malignant melanoma cell line) cells labeling CD63 with ab252919 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing lysosome/endosomes co-staining in SK-MEL-28 cell line. The nuclear counterstain is DAPI (blue). Lysosomes/endosomes are detected with Anti-LAMP1 antibody [H4A3] (ab25630) at 1/200 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.