重组Anti-PAX6抗体[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.

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.

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.

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 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.