Anti-Frizzled 6 抗体 [EPR25319-149] (ab290728)

Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS was used as a blocking and diluting buffer and concentration. 

Samples are non-boiled as boiling may cause protein aggregates.
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
False colour image of Western blot: Anti-FZD6 antibody [EPR25319-149] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab290728 was shown to bind specifically to FZD6. A band was observed at 79 kDa in wild-type Hela cell lysates with no signal observed at this size in FZD6 knockout cell line ab265280 (knockout cell lysate ab258874). To generate this image, wild-type and FZD6 knockout Hela cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/10000 dilution.

Low expression: Daudi, Raji, K562 (PMID: 9480858).
Samples are non-boiled as boiling may cause protein aggregates.
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
False colour image of Western blot: Anti-FZD6 antibody [EPR25319-149] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 loading control staining at 1/20000 dilution, shown in red.
First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/10000 dilution.

Immunohistochemical analysis of paraffin-embedded human cervical carcinoma tissue labelling Frizzled 6 with ab290728 at 1/100, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human cervical carcinoma.
The section was incubated with ab290728 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection). 

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

Immunohistochemical analysis of paraffin-embedded human endometrial carcinoma tissue labelling Frizzled 6 with ab290728 at 1/100, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human endometrial carcinoma.
The section was incubated with ab290728 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection). 

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

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HeLa (human cervix adenocarcinoma epithelial cell) cell pellet
(B) FZD6 knockout HeLa (ab265280) cell pellet labelling Frizzled 6 with ab290728 at 1/200, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human endometrial carcinoma.
The section was incubated with ab290728 for 30 mins at room temperature. Cytoplasmic staining on (A) Wild-type HeLa (human cervix adenocarcinoma epithelial cell) cell pellet (ab255928), no staining on (B) FZD6 knockout HeLa (ab265280) cell pellet.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection). 

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