Anti-CD63 antibody [MX-49.129.5]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD63 antibody [MX-49.129.5] (AB193349)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SK-MEL-28 (Human malignant melanoma) cells labelling CD63 with ab193349 at 1/50 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in SK-MEL-28 cell line. The nuclear counter stain is DAPI (blue).

CD63 is also detected with Rabbit monoclonal Anti-CD63 antibody [EPR22458-280] (ab252919) at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) (ab150080) secondary antibody at 1/500 dilution (red).
The negative controls are as follows :
-ve control 1 : ab193349 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) (ab150080) secondary antibody at 1/500 dilution.
-ve control 2 : Anti-CD63 antibody [EPR22458-280] (ab252919) at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) (Alexa Fluor^® 488) (ab150113) secondary antibody at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [MX-49.129.5] (AB193349)

Immunohistochemical analysis of paraffin-embedded human melanoma tissue labelling CD63 with ab193349 at 1/5000 dilution, followed by Goat Anti-Mouse IgG H&L (HRP polymer) (ab214879) at the supplied dilution. Cytoplasmic staining on human melanoma is observed. Counter stained with Hematoxylin. Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) was performed for 20 mins, before the section was incubated with ab193349 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Secondary antibody only control : Used PBS instead of primary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [MX-49.129.5] (AB193349)

Negative control staining using ab193349. Immunohistochemical analysis of paraffin-embedded human breast tissue labelling CD63 with ab193349 at 1/5000 dilution, followed by Goat Anti-Mouse IgG H&L (HRP polymer) (ab214879) at the supplied dilution. No staining on human breast is observed, as expected (PMID : 22957045). Counter stained with Hematoxylin. Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) was performed for 20 mins, before the section was incubated with ab193349 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• Flow Cyt

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Flow Cytometry - Anti-CD63 antibody [MX-49.129.5] (AB193349)

Flow cytometric analysis of SK-MEL-28 (Human malignant melanoma) cell line labelling CD63 with ab193349 at 1/1000 dilution (red) compared with a Mouse monoclonal IgG (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488, ab150113) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CD63 antibody [MX-49.129.5] (AB193349)

Flow cytometric (intracellular) analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilised SK-MEL-28 (Human malignant melanoma) cell line labelling CD63 with ab193349 at 1/1000 dilution (red) compared with a Mouse monoclonal IgG (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488, ab150113) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt

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Flow Cytometry - Anti-CD63 antibody [MX-49.129.5] (AB193349)

Flow cytometric analysis of human peripheral blood mononuclear cells (PBMC) labeling CD63 with ab193349 at 1/1000 dilution (right), compared with a Mouse monoclonal IgG (left). Goat anti mouse IgG (Alexa Fluor^® 488, ab150113), at 1/2000 dilution was used as the secondary antibody.

Cells were stained with mouse IgG or ab193349, then stained with anti-CD14 conjugated to Alexa Fluor^® 647. Gated on viable cells.

• WB

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Western blot - Anti-CD63 antibody [MX-49.129.5] (AB193349)

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

CD63 can undergo glycosylation as shown in lane 1 and 2 (PMID : 9890706, 28740179).
Samples are non-boiled as boiling may cause protein aggregates.

All lanes:

Western blot - Anti-CD63 antibody [MX-49.129.5] (ab193349) at 1/1000 dilution

Lane 1:

HUVEC (human umbilical vein endothelial cell), whole cell lysate at 40 µg

Lane 2:

HL-60 (human Acute Promyelocytic Leukemia promyeloblast), whole cell lysate at 40 µg

Secondary

All lanes:

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

false

Exposure time: 59s