Anti-PAX6 antibody [EPR15858]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR15858] (AB195045)

IHC image of Pax6 staining in a formalin fixed, paraffin embedded normal human cerebellum 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 ab195045, 1/1000 dilution, 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR15858] (AB195045)

Immunohistochemical analysis of paraffin-embedded Human retinoblastoma tissue labeling PAX6 with ab195045 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab97051) at 1/500 dilution. Nuclear staining on human retinoblastoma tissue is observed. Counter stained with Hematoxylin.

Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR15858] (AB195045)

Immunohistochemical analysis of paraffin-embedded Human retina tissue labeling PAX6 with ab195045 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab97051) at 1/500 dilution. Nuclear staining on human retina tissue is observed. Counter stained with Hematoxylin.

Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR15858] (AB195045)

Immunohistochemical analysis of formalin fixed paraffin embedded human cerebrum labelling PAX6 with ab195045 at a concentration of 1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab195045 Anti-PAX6 antibody [EPR15858] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR15858] (AB195045)

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling PAX6 with ab195045 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab97051) at 1/500 dilution. Nuclear staining on human pancreas tissue is observed. Counter stained with Hematoxylin.

Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PAX6 antibody [EPR15858] (AB195045)

Immunofluorescence analysis of 4% paraformaldehyde-fixed, 0.1% tritonX-100 Y79 (Human retinoblastoma cell line) cells labeling PAX6 with ab195045 at 1/350, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 (green).

Confocal image showing cytoplasmic and nuclear staining on Y79 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 (red).

The negative controls are as follows : -
-ve control 1 : ab195045 at 1/350 followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500.

-ve control 2 : ab7291 at 1/500 followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAX6 antibody [EPR15858] (AB195045)

IHC image of Pax6 staining in a formalin fixed, paraffin embedded normal rat cerebellum 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 ab195045, 1/1000 dilution, 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PAX6 antibody [EPR15858] (AB195045)

ab195045 staining Pax6 in primary mouse neurons/glia, DIV1 (top row) and DIV14 (bottom row) both prepared from E18 mouse hippocampal brain area (obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. C57EHP). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab195045 at 0.2 µg/ml and ab7291, mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue). A subset of cells showed staining in the nucleus at DIV1 (likely neuroprogenitor cells), while differentiated neurons (DIV14) where Pax6 negative. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PAX6 antibody [EPR15858] (AB195045)

Immunofluorescence analysis of 4% paraformaldehyde-fixed, 0.1% tritonX-100 Neuro-2a (mouse neuroblastoma) cells labeling PAX6 with ab195045 at 1/350, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 (green).

Confocal image showing cytoplasmic and nuclear staining on Neuro-2a cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 (red).

The negative controls are as follows : -

-ve control 1 : ab195045 at 1/350 followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500.

-ve control 2 : ab7291 at 1/500 followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400.

• WB

Supplier Data

Western blot - Anti-PAX6 antibody [EPR15858] (AB195045)

Blocking/Dilution buffer : 5% NFDM/TBST.

The 47kDa band represents the full length PAX6, the 32kDa & 33kDa bands represent the PAX6p32 & PAX6p33 fragments (PMID : 21084637, PMID : 32827359).

All lanes:

Western blot - Anti-PAX6 antibody [EPR15858] (ab195045) at 1/10000 dilution

Lane 1:

Y79 cell lysate at 20 µg

Lane 2:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 47 kDa

Observed band size: 32 kDa,33 kDa,47 kDa

false

• WB

Lab

Western blot - Anti-PAX6 antibody [EPR15858] (AB195045)

Western blot : Anti-PAX6 antibody [EPR15858] (ab195045) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab195045 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 [EPR15858] (ab195045) at 1/1000 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

false

• WB

Supplier Data

Western blot - Anti-PAX6 antibody [EPR15858] (AB195045)

Blocking/Dilution buffer : 5% NFDM/TBST.

The 47kDa band represents the full length PAX6, the 32kDa & 33kDa bands represent the PAX6p32 & PAX6p33 fragments (PMID : 21084637, PMID : 32827359).

All lanes:

Western blot - Anti-PAX6 antibody [EPR15858] (ab195045) at 1/1000 dilution

All lanes:

Mouse eyeball lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 47 kDa

Observed band size: 32 kDa,33 kDa,47 kDa

false

• WB

Supplier Data

Western blot - Anti-PAX6 antibody [EPR15858] (AB195045)

Blocking/Dilution buffer : 5% NFDM/TBST.

The transfected lysate spans the immunogenic region : aa1-aa422 (47kDa).

All lanes:

Western blot - Anti-PAX6 antibody [EPR15858] (ab195045) at 1/10000 dilution

Lane 1:

PAX6 transfected 293T cell lysate at 10 µg

Lane 2:

Non-transfected 293T cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 47 kDa

Observed band size: 32 kDa,33 kDa,47 kDa

false