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Sasha Bakhru: Immunofluorescence Staining of NSCs

For the purpose of probing cell phenotype beyond culture, immuno?uorescence staining of paraformaldehyde-?xed cells was employed. Cells attached to PLO/LNcoated substrates were ?xed in 4% paraformaldehydePBS solution (Sigma) for thirty minutes.

Following ?xation, NSCs were washed 3 times in PBS (Gibco, Grand Island, NY)) and subjected to 60 minutes of blocking and permeabilization in TBS (BioRad, Hercules, CA)) with 5% donkey serum (Sigma) and 0.25% Triton X100 (Sigma) (TBS++).

NSCs were then incubated with combinations of one mouse and one rabbit primary antibody, of msNestin, rbTuj1, rbGFAP, rbKI67, in TBS++ overnight at 4C. The following day, NSCs were washed 3 times with TBS and incubated for 90 minutes with ?uorescently labeled secondary antibodies (rbCy2, msCy3) (Peprotech, Rocky Hill, NJ).

Cells were washed 3 more times with TBS and subject to nuclear staining with DAPI (Roche, Nutley, NJ) in PBS for 5 minutes.

After 3 final washes in PBS, cells were then imaged with a Nikon TE2000 LSCM.