Anti-PAX6 抗体 [EPR3352(2)]

• mIHC

Lab

Multiplex immunohistochemistry - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded Human retina tissue labeling PAX6, Glutamine Synthetase and CRX with ab109233 at 1/10000 dilution, ab240193 at 1/20000 dilution and ab248897 at 1/1000 dilution followed by a ready to use Opal Polymer HRP Ms + Rb secondary antibody. Nuclear counter stain used was DAPI. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins Panel A : merged staining of anti-CRX (gray; Opal™690), anti-Glutamine Synthetase (green; Opal™520) and anti-PAX6 (red; Opal™570) on human retina. Panel B : anti-PAX6 stained on retinal progenitor cells. Panel C : anti-Glutamine Synthetase stained on Müller glia. Panel D : anti-CRX stained on subset cells of outer nuclear layer and inner nuclear layer. The section was incubated in three rounds of staining : in the order of ab248897, ab240193, and ab109233 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse retina tissue staining RPE65 with ab231782 at a 1/8000 dilution, ab109233 anti-PAX6 used at 1/1000 dilution and ab221664 anti-RHO used at a 1/30000 dilution.

Panel A : merged staining of anti-RPE65 (green; Opal™520), anti-PAX6 (magenta; Opal™690) and anti-RHO (gray; Opal™570) on mouse retina.

Panel B : anti-RPE65 staining pigmented layer in mouse retina.

Panel C : ant-PAX6 staining inner nuclear layer and ganglion cell layer in mouse retina.

Panel D : ant-RHO staining rod-shaped photoreceptor cells in mouse retina.

Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab231782, ab109233 and ab221664 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

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

• WB

Lab

Western blot - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

Western blot : Anti-PAX6 antibody [EPR3352(2)] (ab109233) staining at 1/5000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab109233 was shown to bind specifically to PAX6. A band was observed at 50 kDa in wild-type A549 cell lysates with no signal observed at this size in PAX6 knockout cell line. To generate this image, wild-type and PAX6 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-PAX6 antibody [EPR3352(2)] (ab109233) at 1/5000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

PAX6 knockout A549 cell lysate at 20 µg

Lane 3:

HEK-293 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 50 kDa

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• WB

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Western blot - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

All lanes:

Western blot - Anti-PAX6 antibody [EPR3352(2)] (ab109233) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

K562 cell lysate at 10 µg

Lane 3:

HepG2 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 47 kDa

Observed band size: 47 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat pancreas tissue sections labeling PAX6 with Purified ab109233 at 1 : 2000 dilution (0.52 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

IHC image of Pax6 staining in normal rat retina formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab109233, 1μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

IHC image of Pax6 staining in normal mouse retina formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab109233, 1μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse retina tissue sections labeling PAX6 with Purified ab109233 at 1 : 2000 dilution (0.52 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human retina tissue sections labeling PAX6 with Purified ab109233 at 1 : 2000 dilution (0.52 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human pancreas tissue sections labeling PAX6 with Purified ab109233 at 1 : 2000 dilution (0.52 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use). Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

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Immunoprecipitation - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

ab109233 (purified) at 1/50 dilution (20μg/ml) immunoprecipitating PAX6 in HeLa whole cell lysate.

Lane 1 (input) : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab109233 & HeLa whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109233 in HeLa whole cell lysate

For western blotting, ab109233 at 1/500 and veriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.

Blocking and diluting buffer : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-PAX6 antibody [EPR3352(2)] (ab109233)

Predicted band size: 47 kDa

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• WB

Unknown

Western blot - Anti-PAX6 antibody [EPR3352(2)] (AB109233)

All lanes:

Western blot - Anti-PAX6 antibody [EPR3352(2)] (ab109233) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

Mouse retina lyates at 15 µg

Lane 3:

Rat retina lyates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 47 kDa

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