Tiling microscope

A subset of larvae (stages 30-34) that were processed by ISH expression were embedded in a gelatin-based medium [0.5% gelatin, 30% bovine serum albumin, 20% sucrose, hardened with glutaraldehyde (75 µl/ml)], and vibratome sectioned at 40 µm in the transverse plane. Serial section images through the otic vesicle of a minimum of six embryos per mRNA injection were collected using a Leica Tiling microscope. The area of both control and injected otic vesicles from every section per embryo was measured using Zen Blue software (Zeiss Zen 2.0) to calculate the volume. Because the larvae were of different sizes, the volume of each mutant otic vesicle was expressed as a percentage of the control vesicle of the same larva and plotted as percent change from control: [(experimental−control)/control]×100. A two-tailed paired Student's t-test was used to determine if the volume of the Six1-mutant-injected otic vesicle was significantly different from that on the control side (P<0.05). A two-tailed unpaired Student's t-test was used to determine if the volumes of Six1-mutant-injected otic vesicles were significantly different from those of Six1WT-injected otic vesicles (P<0.05).

Cells were counted in images of sections from each ganglion collected at 20× magnification on a Leica Tiling microscope (embryonic CNgV) or a Leica TCS SP8 multiphoton scanning confocal microscope (P8 CNgV). Red (Six1), green (Wnt1^Cre/Rosa26-GNZ; abbreviated as Wnt1^Cre throughout the text), blue (DAPI) and infrared (Alexa-Fluor 647) channels were visualized separately and superimposed as composite images (Wnt1^Cre::Rosa26-GNZ signal was amplified for imaging using anti-eGFP antibody labeling). Labeled cells were counted as previously described (Karpinski et al., 2016 (link)). To determine proportions of proliferating cells or differentiating neurons in embryonic material, numbers of cells positive for BrdU or NeuN were assessed for Six1, Wnt1^Cre(GFP), Six1/ Wnt1^Cre or DAPI labeling, and percentages were calculated. To assess TrkB, TrkA, Ret or TrpV1-labeled neuron subpopulations, fields were counted for all βIII-tubulin⁺ neurons, followed by those co-labeled for TrkB/TrkA/Ret/Trpv1, and percentages were determined. Chi-Square/Fisher exact tests, t-tests or ANOVA were used to assess statistical differences between genotypes and cell classes as described for each experiment in the Results.