Etchevers:Notebook/Genomics of hNCC/2008/11/26

Genomics of human neural crest cells

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Corneas and cells Immuno to see if melanoblasts in cell cultures: 2 drops of ImageIT-Fx was applied to each at RT; I think I had left them in blocking solution this whole time. Primary antibodies were CNPase, IgG1, and tyrosinase, IgG2a. They were used at a dilution of 10 μg in 580 μL and incubated for 2h from 9:15-11:15 at room temperature under agitation. 4 × 30-min washes PBT. Secondary antibodies conjugated to Alexa Fluor fluorophores were used at a 1:100 dilution and were incubated overnight in the dark in the fridge. Used Alexa 633 - IgG1 and Alexa 555 - IgG2. Tomorrow: 3 × 5-min washes PBT. Coverslips mounted with ProLong Gold mounting media containing DAPI. The rabbit corneas had some hNCC still in them. Took lots of photos, will need to also look in confocal and do double-staining now on adjacent sections with PGD2 and/or beta-2-microglobulin. Encouraging! Heather 11:11, 26 November 2008 (EST):