Anti-Frizzled 6 antibody [EPR25319-149]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Frizzled 6 antibody [EPR25319-149] (AB290728)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HeLa ( tissue labeling Frizzled 6 with ab290728 at 1/200 (2.47 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. 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 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 : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Frizzled 6 antibody [EPR25319-149] (AB290728)

Immunohistochemical analysis of paraffin-embedded Human endometrial ca tissue labeling Frizzled 6 with ab290728 at 1/100 (4.94 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. 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 : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Frizzled 6 antibody [EPR25319-149] (AB290728)

Immunohistochemical analysis of paraffin-embedded Human cervical carci tissue labeling Frizzled 6 with ab290728 at 1/100 (4.94 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. 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 : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• WB

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Western blot - Anti-Frizzled 6 antibody [EPR25319-149] (AB290728)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBSSamples 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.

All lanes:

Western blot - Anti-Frizzled 6 antibody [EPR25319-149] (ab290728) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 20 µg

Lane 2:

FZD6 knockout HeLa (<a href='/en-us/products/cell-lines/human-fzd6-frizzled-6-knockout-hela-cell-line-ab265280'>ab265280</a>) whole cell lysate 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Observed band size: 79 kDa

false

• WB

Supplier Data

Western blot - Anti-Frizzled 6 antibody [EPR25319-149] (AB290728)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBSLow 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.

All lanes:

Western blot - Anti-Frizzled 6 antibody [EPR25319-149] (ab290728) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 20 µg

Lane 2:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate 20 µg

Lane 3:

K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 20 µg

Lane 4:

Daudi (human Burkitts lymphoma lymphoblast) whole cell lysate 20 µg

Lane 5:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate 20 µg

Secondary

Lanes 1 - 5:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Lanes 1 - 5:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Observed band size: 79 kDa

false