Anti-beta III Tubulin antibody - Neuronal Marker (ab18207)

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Mice were sacrificed and corneal whole mounts were prepared as described previously [14]. Corneas were excised and directly fixed in 4% paraformaldehyde (PFA) overnight at 4°C. The corneas were washed thrice with PBS for 5 min each at room temperature. This was followed by permeabilization of the corneas in PBS containing 3% NP40, 3% BSA, 0.25% gelatin, 5mM EDTA for 12 h at room temperature. The corneas were then washed thrice with PBS for 5 min each at room temperature. The corneas were then blocked in Blocking solution (PBS containing 0.025% NP40, 1% BSA and 2.5% Donkey Serum) overnight at 4°C. Furthermore, the corneas were incubated with primary antibody at appropriate dilution in blocking solution overnight at 4°C followed by 3 washes with PBS for 5 min each at room temperature. Fluorescence-coupled secondary antibodies in blocking solution at appropriate dilutions were then added to the corneas and incubated overnight at 4°C. The corneas were then washed with PBS thrice for 5 min each at room temperature. Corneas were mounted onto glass slides with a drop of 4', 6-diamidino-2-phenylindole (DAPI)-containing mounting medium and covered with a coverslip.

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