All lanes : Anti-CD63 antibody [KILL150A] (ab271286) at 0.978 µg/ml

Lane 1 : SK-MEL-28 (human malignant melanoma), whole cell lysate
Lane 2 : HUVEC (human umbilical vein endothelial cell), whole cell lysate
Lane 3 : Human lymph node tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 26 kDa

CD63 can undergo glycosylation as shown in lane 1, 2 and 3 (PMID: 9890706, 28740179).

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-2: 3 mins; Lane 3: 48 secs.

This blot was developed using a higher sensitivity ECL substrate.

Immunohistochemical analysis of paraffin-embedded human melanoma tissue labeling CD63 with ab271286 at 1/4000 dilution (0.978µg/ml) followed by ready to use Goat Anti-mouse IgG H&L (HRP polymer) (ab214879). Cytoplasmic staining on human melanoma. The section was incubated with ab271268 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Goat Anti-mouse IgG H&L (HRP polymer) (ab214879).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling CD63 with ab271286 at 1/4000 dilution (0.978µg/ml) followed by ready to use Goat Anti-mouse IgG H&L (HRP polymer) (ab214879). The section was incubated with ab271268 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Negative control: Nearly no staining on human breast.

Secondary antibody only control: Secondary antibody is ready to use Goat Anti-mouse IgG H&L (HRP polymer) (ab214879).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SK-MEL-28 cells labelling CD63 with ab271286 at 1/50 dilution (19.56µg/ml), followed by ab150113 Goat Anti-mouse IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing lysosome/endosomes (ab252919) co-staining in SK-MEL-28 cells.

ab252919 Anti-CD63 rabbit monoclonal antibody was used to counterstain tubulin at 1/250 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at a 1/1000 dilution (Red). The nuclear counterstain was DAPI (Blue).

Negative control 1: ab271286 at a 1/50 dilution (19.56µg/ml) followed by ab150080 at a 1/1000 dilution.

Negative control 2: ab259919 at a 1/200 dilution followed by ab150113 at a 1/1000 dilution.

Flow cytometric analysis of NK-92 (human malignant non-Hodgkin's lymphoma natural killer cell) cells labelling CD63 with ab271286 at 1/1000 dilution (19.56µg/ml) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.

Flow cytometric analysis of SK-MEL-28 (human malignant melanoma) cells labelling CD63 with ab271286 at 1/1000 dilution (19.56µg/ml) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.