Detail Immunostaining Protocol for Pax6 mouse eye samples

Detail Immunostaining Protocol for Pax6 mouse eye samples

(Djalilian’s Lab, Ophthalmology and Visual Science, UIC)

1, Pax6 mouse scarified according to animal protocol. Mouse eyes were enucleated and washed in PBS solution.

2, For whole mount staining: corneas were dissected and fixed with 4% paraformaldehyde at 4°C overnight. On the next day, wash the corneas with PBS, they were incubated with proteinase K (20ug/ml: sigma) for 5 min at room temperature followed by 100% methanol for another 30min.

3, For cryo-section staining: mouse eye were put in OCT compound and cut to 10um thickness. Section were fixed for 10min in chilled 4% paraformaldehyde.

4, For staining, all the samples were blocked at room temperature with blocking buffer (10% donkey serum) for 1 hour.

5, The samples were incubated overnight at 4°C with primary antibody in blocking buffer. For negative controls, the samples were incubated without the primary antibody in blocking buffer.

6, After primary antibody, the samples were washed with TBST 3X, the secondary antibody in blocking buffer was applied for 1 hour at room temperature followed by 3X TBST wash.

7, Whole mount corneal were mounted at P90; cryo-sections were mounted at P30 with DAPI.

8, Images were performed with confocal microscope.