Anti-CD63 antibody [RM1095]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [RM1095] (AB315108)

Immunohistochemical analysis of paraffin-embedded Human breast tissue labeling CD63 with ab315108 at 1/5000 (0.105 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : no staining on the breast epithelial cells, positive staining on the stromal immune cells in human breast. The section was incubated with ab315108 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 a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [RM1095] (AB315108)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling CD63 with ab315108 at 1/5000 (0.105 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : no staining on human skeletal muscle cells, but weak staining on the immune cells. The section was incubated with ab315108 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 a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [RM1095] (AB315108)

Immunohistochemical analysis of paraffin-embedded Human melanoma tissue labeling CD63 with ab315108 at 1/5000 (0.105 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human melanoma. The section was incubated with ab315108 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 a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [RM1095] (AB315108)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CD63 with ab315108 at 1/5000 (0.105 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil. The section was incubated with ab315108 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 a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• Flow Cyt

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Flow Cytometry - Anti-CD63 antibody [RM1095] (AB315108)

Flow cytometric analysis of Jurkat (human T cell leukemia T lymphocyte from peripheral blood, Left) / SK-MEL-28 (human malignant melanoma cell, Right) cells labelling CD63 with ab315108 at 1/500 dilution (0.1 ug)/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, ab150081) at 1/5000 dilution was used as the secondary antibody.

Low expression : Jurkat. Gated on viable cells.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD63 antibody [RM1095] (AB315108)

Immunofluorescent analysis of 80% Methanol-fixed, 0.1% TritonX-100 permeabilized SK-MEL-28 (human malignant melanoma) cells labelling CD63 with ab315108 at 1/100 (5.24 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining in SK-MEL-28 cell line. Low expression cell line : Jurkat. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution.

• IP

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Immunoprecipitation - Anti-CD63 antibody [RM1095] (AB315108)

CD63 was immunoprecipitated from 0.35 mg SK-MEL-28 (human malignant melanoma cell ) whole cell lysate with ab315108 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315108 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : SK-MEL-28 (human malignant melanoma cell ) whole cell lysate

Lane 2 : ab315108 IP in SK-MEL-28 (human malignant melanoma cell ) whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab315108 in SK-MEL-28 whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-CD63 antibody [RM1095] (ab315108) at 1/30 dilution

All lanes:

SK-MEL-28 (human malignant melanoma cell ) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 180s

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD63 antibody [RM1095] (AB315108)

Immunofluorescent analysis of 80% Methanol-fixed, 0.1% TritonX-100 permeabilized C2C12 (mouse myoblasts myoblast) cells labelling CD63 with ab315108 at 1/100 (5.24 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining in C2C12 cells. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution.

• WB

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Western blot - Anti-CD63 antibody [RM1095] (AB315108)

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

Low expression : Jurkat.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-CD63 antibody [RM1095] (ab315108) at 1/1000 dilution

Lane 1:

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

Lane 2:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 3:

bEnd.3 (mouse brain endothelial cell) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 5:

C2C12 (mouse myoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 30-65 kDa,36 kDa

false

Exposure time: 180s

• WB

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Western blot - Anti-CD63 antibody [RM1095] (AB315108)

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

CD63 is a glycoprotein of approximately 30-65 kDa and detected as 25kDa bands after de-glycosylation treatment.

Exposure time : Lanes 1-2 : 37 seconds; Lanes 3-4 : 180 seconds

All lanes:

Western blot - Anti-CD63 antibody [RM1095] (ab315108) at 1/1000 dilution

Lane 1:

Untreated SK-MEL-28 (human malignant melanoma cell) whole cell lysate at 15 µg

Lane 2:

SK-MEL-28 whole cell lysate treated with Peptide:N-glycosidase F (PNGase F) at 15 µg

Lane 3:

Untreated bEnd.3 (mouse brain endothelial cell) whole cell lysate at 15 µg

Lane 4:

bEnd.3 whole cell lysate treated with Peptide:N-glycosidase F (PNGase F) at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 30-65 kDa,25 kDa

false