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2 protocols using goat anti sox2 sc 17320

1

Cochlear Sensory Epithelium Immunostaining

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5 days after injection, the mice were sacrificed by CO2 inhalation, and the cochleae were fixed with 4% (weight/volume) paraformaldehyde (Sigma) at 4°C overnight and then decalcified in 10% EDTA (Sigma) for 3 days if necessary. Tissues were permeabilized with 0.3% Triton X-100 (1% PBS-Tween 20 [PBS-T]) and blocked with 10% donkey serum for 1 h at room temperature. All primary and secondary antibodies were diluted in 1% PBS-T. To observe the tdT expression in IHCs and OHCs of the cochlear and utricle sensory epithelium, we used 1:600 rabbit anti-myosin (Myo)7a (#25-6790; Proteus BioSciences, Ramona, CA, USA) and 1:300 goat anti-Sox2 (sc-17320; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. Appropriate Alexa-conjugated secondary antibodies (A21206 and A11058; Invitrogen, Gaithersburg, MD, USA) were incubated for 1 h after three rinses with PBS rinses, and DAPI was used to label the nuclei (1:1,000 dilution; Sigma).
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2

Immunofluorescence Staining of Stem Cells

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For immunofluorescence, cells were fixed for 20 minutes with 4% paraformaldehyde (EM Sciences) in PBS. Permeabilization and blocking were performed in a single step using PBS (Lonza) supplemented with 0.1% Triton X-100 (Sigma), 10% FCS (GE Healthcare) and 1% BSA. Primary antibodies were applied overnight at 4°C in PBS containing 0.1% BSA. Subsequently, cells were incubated with secondary antibodies for 1 h at room temperature and ultimately washed 3x, including a Hoechst (Thermo Fisher Scientific) counterstaining for nuclei in the second washing step. Primary antibodies used in this study included: goat anti-Sox2 sc-17320 1:200 and mouse anti-Oct4 sc-5279 1:200 (both Santa Cruz), mouse anti-SSEA4 MC-813-70 1:200 (DSHB), goat anti-Sox1 AF3369 1:400, mouse anti-Nestin MAB1259 1:500, (both R&D Systems), rabbit anti-TH sc-14007 1:300 (Santa Cruz), mouse anti-Tuj1 MMS-435P 1:1000 (BioLegend). For fluorescence microscopy analysis, secondary antibodies conjugated to Alexa-488, 568 or 647 (1:1000, Thermo Fisher Scientific) were used. Cells were imaged on an inverted Apotome Zeiss Axio/Observer Z1 fluorescence microscope.
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