ab78545 staining PAX6 in Neuro2a cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% 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 with ab78545 at 1/50 dilution and ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

IHC image of Pax6 staining in a formalin fixed, paraffin embedded normal rat retina 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 ab78545, 5µg/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 a formalin fixed, paraffin embedded normal human pancreas 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 ab78545, 20µg/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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

IHC image of PAX6 staining in a section of frozen normal human pancreas performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab78545, 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.

Immunohistochemical analysis of PFA-fixed frozen murine embryonic brain coronal sections, labelling PAX6 with ab78545 at a dilution of 1/100 incubated for 8 hours at 4°C in blocking buffer diluent. Permeabilization was with Triton X-100 and blocking was with 1% serum for 1 hour. Heat mediated antigen retrival was with 10mM sodium citrate buffer pH6.0 for 10 minutes at 650W in a microwave. The secondary was a goat Alexa Fluor^® 488 at 1/700.

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IHC image of PAX6 staining in mouse e14 foetus formalin fixed paraffin embedded tissue section, with the use of Mouse on Mouse Polymer IHC Kit (Ab127055). The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab78545, 10µg/ml overnight at +4°C. The Mouse on Mouse HRP Polymer was incubated for 15 minutes at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

Immunohistochemical analysis of mouse brain tissue, staining PAX6 with ab78545.

Tissue was fixed with paraformaldehyde and blocked with 10% serum for 1 hour at room temperature. Samples were incubated with primary antibody (1/100 in PBST) for 12 hours at 4°C. An AlexaFluor®568-conjugated goat anti-mouse polyclonal IgG (1/1000) was used as the secondary antibody.

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