Anti-CD63 antibody [EPR5702] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [EPR5702] - BSA and Azide free (AB215821)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD63 with purified ab134045 at 1/2000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134045).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [EPR5702] - BSA and Azide free (AB215821)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle tissue (negative control) labelling CD63 with purified ab134045 at 1/2000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134045).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [EPR5702] - BSA and Azide free (AB215821)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human melanoma tissue labelling CD63 with unpurified ab134045 at a dilution of 1/250.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134045).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD63 antibody [EPR5702] - BSA and Azide free (AB215821)

This data was developed using the same antibody clone in a different buffer formulation (ab134045).

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil staining CD63 with ab134045 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab134045 anti-CD63 [EPR5702] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• WB

Lab

Western blot - Anti-CD63 antibody [EPR5702] - BSA and Azide free (AB215821)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134045).

Lanes 1 - 6 : Merged signal (red and green). Green - ab134045 observed at 26 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab134045 was shown to specifically react with CD63 in wild-type HAP1 Brefeldin A treated cells as signal was lost in HAP1 Brefeldin A treated CD63 knockout cells. Wild-type and CD63 knockout samples were subjected to SDS-PAGE. ab134045 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CD63 antibody [EPR5702] - BSA and Azide free (ab215821) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Brefeldin A treated wild-type HAP1 whole cell lysate at 20 µg

Lane 3:

CD63 knockout HAP1 whole cell lysate at 20 µg

Lane 4:

Brefeldin A treated CD63 knockout HAP1 whole cell lysate at 20 µg

Lane 5:

HL60 whole cell lysate at 20 µg

Lane 6:

Human platelets whole cell lysate at 20 µg

Predicted band size: 26 kDa

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• WB

Lab

Western blot - Anti-CD63 antibody [EPR5702] - BSA and Azide free (AB215821)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134045). Anti-CD63 antibody [EPR5702] (ab134045) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab134045 was shown to bind specifically to CD63. A band was observed at 30 kDa in wild-type HepG2 cell lysates with no signal observed at this size in CD63 knockout cell line ab280796 (knockout cell lysate ab283828). To generate this image, wild-type and CD63 knockout HepG2 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CD63 antibody [EPR5702] - Late Endosome Marker (<a href='/en-us/products/primary-antibodies/cd63-antibody-epr5702-late-endosome-marker-ab134045'>ab134045</a>) at 1/1000 dilution

Lanes 1 - 4:

Western blot at 20 µg

Lanes 3 - 4:

Western blot - Human CD63 knockout Hep G2 cell lysate (ab283828)

Lanes 3 - 4:

Western blot - Human CD63 knockout Hep G2 cell line (<a href='/en-us/products/cell-lines/human-cd63-knockout-hep-g2-cell-line-ab280796'>ab280796</a>)

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 26 kDa

Observed band size: 30 kDa

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