Poly28400

Enteric neurospheres or their dispersed forms grown on fibronectin-coated coverslips in SRM-filled wells were first treated with BD Cytofix/Cytoperm™ Kit. Then, specimens were incubated with primary antibodies against the target proteins of vimentin (Poly29191, BioLegend), TUBB3 (TUJ1, BioLegend), glial fibrillary acidic protein (GFAP, Poly28400, BioLegend), p75 (Clone 25-8, LSBio), neuronal nitric oxide synthase (nNOS, Cat No: ab5586, Abcam) or choline acetyltransferase (ChAT, Cat No: ab34419, Abcam), followed by fluorescence-conjugated secondary antibodies against the species the primary antibodies were raised in. Fluorescence-conjugated anti-S100b or tyrosine hydroxylase (TH, Clone 2/40/15, BioLegend) were added to specimens after the completion of secondary antibody reaction and washing. Counterstain for the nuclei was performed using diamidino-2-phenylindole (DAPI). After washing, specimens were mounted with Dako fluorescence mounting medium and examined under a Leica confocal microscope.

Cells grown on coverslips were first subjected to fixation and permeabilization with BD Cytofix/Cytoperm^TM Kit. Then, samples were incubated with primary antibodies against TUBB3 and glial fibrillary acidic protein (GFAP, Poly28400, BioLegend), followed by fluorescence-conjugated secondary antibodies of rat anti-mouse IgG_2a (BioLegend) and donkey anti-rabbit IgG (BioLegend). Counterstain for the nuclei was performed using diamidino-2-phenylindole (DAPI, Sigma-Aldrich). After washing, specimens were mounted with Dako fluorescence mounting medium (Agilent, Santa Clara, USA) and examined under a Leica confocal microscope.