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Immunofluorescence of H9 cells on day 21 of differentiation for neuronal markers showed >90% of cocultured cells immunostained positive for NCAM (Figure 3(a)) with PRPH positivity in 20% (Figure 3(c)).
The addition of BMP4 (10 ng/ml) on days 5-9 of coculture led to a 12-fold increase in PRPH expression from day 7 to day 21 of differentiation compared to 8-fold without BMP4 (Figure 4(a)).
The sections were then incubated at 4[degrees]C overnight in a combination of the following primary antibodies: (1) mouse anti-tubulin [beta]3 (Tuj1, BioLegend), diluted 1:300; (2) rabbit antiperipherin (Prph, Abcam), diluted 1 : 500.
We immunostained frozen sections of the whole cochleae for two marker proteins: (1) tubulin [beta]3 (Tuj1), which is expressed in both type I and type II SGNs, and (2) peripherin (Prph), a type III intermediate filament, whose immunoreactivity is restricted to the soma and processes of type II SGNs in the mature cochlea.
The variations in the expression of 17 genes (underlined in Table 2) are involved in specific neuronal pathways, such as neuronal cell structure (GPM6B, PRPH, RELN), neural signaling and development (HOXB2, ELAVLI, EFNB1, NRG2, NELL1, MDK, GFRA1), synaptogenesis and steroid biosynthesis (SYCP2, PCDH17, STX1B2, SCG5, Nstage 4 diseasel, GATA3, NROB1).
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