Anti-ZIC3 antibody [EPR30592-522]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZIC3 antibody [EPR30592-522] (AB325172)

Immunohistochemical analysis of paraffin-embedded Mouse E14.5 (brain section) tissue labeling ZIC3 with ab325172 at 1/2000 (0.257 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse E14.5 brain. (A) Low‑powered (magnification, x20) and (B) high‑powered (magnification, x200) microscopic images. (PMID : 17507568).
The primary antibody was incubated 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

• IP

Supplier Data

Immunoprecipitation - Anti-ZIC3 antibody [EPR30592-522] (AB325172)

ZIC3 was immunoprecipitated from 0.35 mg ES-D3 [D3] (mouse blastocyst-derived embryonic stem cell) whole cell lysate with ab325172 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325172 at 1/1000 dilution.

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

To minimize protein degradation, cells were lysed immediately after harvest and then applied for Immunoprecipitation as soon as possible.

All lanes:

Immunoprecipitation - Anti-ZIC3 antibody [EPR30592-522] (ab325172) at 1/1000 dilution

Lane 1:

ES-D3 [D3] (mouse blastocyst-derived embryonic stem cell) whole cell lysate at 10 µg

Lane 2:

ES-D3 [D3] (mouse blastocyst-derived embryonic stem cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325172 in ES-D3 [D3] 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

Observed band size: 50 kDa

false

Exposure time: 41s

• IP

Supplier Data

Immunoprecipitation - Anti-ZIC3 antibody [EPR30592-522] (AB325172)

ZIC3 was immunoprecipitated from 0.35 mg F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate with ab325172 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325172 at 1/1000 dilution.

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

To minimize protein degradation, cells were lysed immediately after harvest and then applied for Immunoprecipitation as soon as possible.

All lanes:

Immunoprecipitation - Anti-ZIC3 antibody [EPR30592-522] (ab325172) at 1/1000 dilution

Lane 1:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325172 in F9 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

Observed band size: 50 kDa

false

Exposure time: 24s

• WB

Supplier Data

Western blot - Anti-ZIC3 antibody [EPR30592-522] (AB325172)

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

Low expression : Neuro-2a; B16-F10

In lanes 1-3, the lysates were stored at -80°C prior to Western Blotting. In lanes 4-7, To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

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

Exposure time : Lanes 1-6 : 26 seconds; Lane 7 : 180 seconds

All lanes:

Western blot - Anti-ZIC3 antibody [EPR30592-522] (ab325172) at 1/1000 dilution

Lane 1:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 3:

B16-F0 (mouse melanoma epithelial-like cell) whole cell lysate at 20 µg

Lane 4:

F9 (mouse embryonal carcinoma epithelial cell) fresh whole cell lysate at 20 µg

Lane 5:

Neuro-2a (mouse neuroblastoma neuroblast) fresh whole cell lysate at 20 µg

Lane 6:

B16-F0 (mouse melanoma epithelial-like cell) fresh whole cell lysate at 20 µg

Lane 7:

ES-D3 [D3] (mouse blastocyst-derived embryonic stem cell) fresh 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 1/20000 dilution

Observed band size: 50 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-ZIC3 antibody [EPR30592-522] (AB325172)

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

Negative control : kidney(PMID : 8557628)

To minimize protein degradation, tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The identity of the bands lower than 50 kDa in lanes 3-4 are unknown.

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

All lanes:

Western blot - Anti-ZIC3 antibody [EPR30592-522] (ab325172) at 1/1000 dilution

Lane 1:

Mouse E12 embryo tissue lysate at 60 µg

Lane 2:

Mouse E15 brain tissue lysate at 60 µg

Lane 3:

Mouse E12-E14.5 brain tissue lysate at 20 µg

Lane 4:

Mouse kidney tissue 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 1/20000 dilution

Observed band size: 50 kDa,36 kDa

false

Exposure time: 59s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZIC3 antibody [EPR30592-522] (AB325172)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling ZIC3 with ab325172 at 1/2000 (0.257 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : No staining on mouse liver (PMID : 8557628).
The primary antibody was incubated 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZIC3 antibody [EPR30592-522] (AB325172)

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling ZIC3 with ab325172 at 1/2000 (0.257 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : No staining on mouse kidney (PMID : 8557628).
The primary antibody was incubated 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

• WB

Supplier Data

Western blot - Anti-ZIC3 antibody [EPR30592-522] (AB325172)

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

This antibody does not cross-react with mouse ZIC1, ZIC2, ZIC4 and ZIC5.

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

In Western blot, Anti-6X His tag® antibody AD1.1.10 staining at 1/1000 dilution.

All lanes:

Western blot - Anti-ZIC3 antibody [EPR30592-522] (ab325172) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a mouse ZIC3 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a mouse ZIC1 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a mouse ZIC2 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a mouse ZIC4 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 6:

293T cells transfected with a mouse ZIC5 expression vector containing a myc-His-tag® 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: 50 kDa,36 kDa

false

Exposure time: 59s