Control and CIH guinea pig CB were perfused and fixed as described in Gonzalez-Obeso et al. (2017) (link). CB were cryoprotected by immersion in 30% (w/v) sucrose in phosphate buffer, embedded individually in Tissue-Tek® (Sakura Finetek, Zoeterwoude, Netherlands) and frozen at -20°C. Tissue sections of 10 μm (Shandon Cryotome, Thermo, Electron Corporation) were collected in glass slices coated with poly L-lysine. CB sections were washed with PBS at room temperature, hematoxylin and eosin stained (H&E, Sigma-Aldrich, MO, United States), dehydrated and mounted with Eukitt Mounting Medium (Merck). Tyrosine hydroxylase (TH) immunofluorescence staining was performed in slices from control and CIH CB, identifying cell nuclei with DAPI. Sections were examined with microscope (Axioscop 2, Zeiss). Images were captured using a digital camera (CoolSNAP, Photometric, Roper Scientific) coupled to the microscope and analyzed using Metamorph 6.3 software.
Dissociated CB cells (Gomez-Niño et al., 2009 (link)) from four different control and four different CIH animals plated on several poly L-lysine-coated coverslips were immunostained for TH and nuclei with DAPI as previously described (Caceres et al., 2007 (link); Gonzalez-Obeso et al., 2017 (link)). Cells were imaged using a laser confocal microscope (LEICA TCS, SP5) and confocal micrographs were processed using LAS software.
Dissociated CB cells (Gomez-Niño et al., 2009 (link)) from four different control and four different CIH animals plated on several poly L-lysine-coated coverslips were immunostained for TH and nuclei with DAPI as previously described (Caceres et al., 2007 (link); Gonzalez-Obeso et al., 2017 (link)). Cells were imaged using a laser confocal microscope (LEICA TCS, SP5) and confocal micrographs were processed using LAS software.
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