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.

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.

All lanes : Anti-PAX6 antibody [EPR15858] (ab195045) at 1/10000 dilution

Lane 1 : PAX6 transfected 293T cell lysate
Lane 2 : Non-transfected 293T cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 47 kDa
Observed band size: 32,33,47 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

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

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 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.

 

Anti-PAX6 antibody [EPR15858] (ab195045) at 1/1000 dilution + Mouse eyeball lysate at 20 µg

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

Predicted band size: 47 kDa
Observed band size: 32,33,47 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

The 47kDa band represents the full length PAX6, we hypothesis the 32kDa & 33kDa bands represent the PAX6p32 & PAX6p33 fragments.

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.

All lanes : Anti-PAX6 antibody [EPR15858] (ab195045) at 1/10000 dilution

Lane 1 : Y79 cell lysate
Lane 2 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 47 kDa
Observed band size: 32,33,47 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

The 47kDa band represents the full length PAX6, we hypothesis the 32kDa & 33kDa bands represent the PAX6p32 & PAX6p33 fragments.

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 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.