Researchers identify important molecularswitch for hearing development NatureComms
. Tissue was embedded in paraffin wax and sectioned to 5-µm, followed by deparaffinization in Roti-Histol and rehydration in a descending ethanol/HO series. For embryonic sections , epitope retrieval was accomplished with citrate-based antigen unmasking solution for 15 min at 100 °C. Postnatal tissue sections were subjected to heat-induced antigen retrieval 10 mM Tris-HCl/2 mM EDTA buffer, pH 9.0, for 90 min at 80 °C.
For sequential double labelling with two primary antibodies generated in the same host, masking of the first primary antibody was performed using the following unconjugated FAB fragments: goat-anti-rabbit FAB fragment , donkey-anti-mouse FAB fragment .Cochlear whole-mounts were rehydrated in a descending methanol/PBS series. For the first permeabilization step, whole-mounts were treated with proteinase K diluted in buffer for 2 min at RT. Digestion was stopped by washing in 0.2% glycine/PBS .
For visualization of NGFR , a biotin-labelled secondary antibody and the TSA Kit were used. Cochlear whole-mounts were incubated with the staining reagent for 30 min at RT, followed by washing with PBS-Triton on ice and inactivation of the horseradish peroxidase by incubation in 6% HFor co-staining of SLC17A8 and F-actin, non-methanol treated cochlear whole-mounts were used and counterstained with Phalloidin-iFluor488 for 1 h at RT.
For transcripts, for which no DNA template was available, primers pairs were designed using the MacVector software , modified with an additional T7-promoter sequence at the 5′ end of the reverse primer, and used for PCR based amplification of DNA templates from genomic DNA or cDNA. Accuracy of all probe templates was confirmed by sequencing.RNA in situ hybridization
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