Anti-CD63 antibody [KILL150A]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CD63 antibody [KILL150A] (AB271286)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [KILL150A] (AB271286)

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.

• Flow Cyt

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Flow Cytometry - Anti-CD63 antibody [KILL150A] (AB271286)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [KILL150A] (AB271286)

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.

• WB

Lab

Western blot - Anti-CD63 antibody [KILL150A] (AB271286)

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.

All lanes:

Western blot - Anti-CD63 antibody [KILL150A] (ab271286) at 1/1000 dilution

Lanes 1 and 5:

Un-boiled HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg

Lanes 2 and 6:

Un-boiled SK-MEL-28 (human malignant melanoma) whole cell lysate at 20 µg

Lane 3:

Boiled HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg

Lane 4:

Boiled SK-MEL-28 (human malignant melanoma) whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Predicted band size: 26 kDa

Observed band size: 30-60 kDa

true

• WB

Lab

Western blot - Anti-CD63 antibody [KILL150A] (AB271286)

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.

All lanes:

Western blot - Anti-CD63 antibody [KILL150A] (ab271286) at 0.978 µg/mL

Lane 1:

SK-MEL-28 (human malignant melanoma), whole cell lysate at 10 µg

Lane 2:

HUVEC (human umbilical vein endothelial cell), whole cell lysate at 10 µg

Lane 3:

Human lymph node tissue lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 26 kDa

false