Dfc420c colour camera

Leaf material was fixed with a solution of 3.7% (w/v) formaldehyde, 5% (v/v) acetic acid, 50% (v/v) ethanol and embedded in LR white. Transverse sections (0.5 μm) were stained with toluidine blue and viewed on a LEICA DM6000 light microscope. The objectives used were ×10/0.4 air, ×20/0.7 air or ×40/0.85 air, and overview images were collected by taking a series of partially overlapping images with a LEICA DFC420C colour camera, which were merged using LEICA LAS‐AF software.

Adult fish (3 mpf; control, n=3; kita-RAS, n=3) were fixed in 4% PFA for 14 days, then decalcified in 1 M EDTA solution for 20 days at room temperature. Larvae (control, n=3; kita-RAS, n=4) were fixed for 2 h. Samples were dehydrated in ethanol and embedded in paraffin, and sagittal sections were taken at 8 µm thickness. Selected slides were de-waxed and stained with Toluidine Blue (Kague et al., 2018 (link)), Alcian Blue, AFOG or Picro-Sirius Red, as performed elsewhere (Hayes et al., 2013 (link)). Images were acquired on a Leica DMI600 inverted microscope, using 20× and 40× oil objectives, LAS software and a DFC420C colour camera. Quantification of thickness of collagen fibre was done using Fiji (Schindelin et al., 2012 (link)), by selecting an area of interest within the bone, followed by measurement of mean intensity of red, blue and green pixels.