Axio a1 compound light microscope

Zebrafish eyes were fixed in 4% paraformaldehyde overnight and then dehydrated in 30% sucrose in phosphate buffer for 24 h. The sucrose was removed and replaced with Optimum Cutting Temperature compound (Fisher Scientific, Toronto, ON, Canada). Eyes were embedded, flash frozen on dry ice, and stored at −80 °C until sectioning. Tissue was then cryosectioned at 10 μm, placed on SuperFrost Plus microscope slides (Fisher Scientific, Toronto, ON, Canada), and stored at −80 °C until use.
In preparation for staining, sections were allowed to thaw at room temperature for 1 h. Tissue was treated with 4% paraformaldehyde for 20 min, washed with water, and rinsed with 60% isopropanol. A working solution of Oil Red O (0.3%) was prepared in 60% isopropanol, and the slides were stained for 15 min in a Coplin jar. The slides were rinsed with 60% isopropanol, and the nuclei were stained with hematoxylin for 1 min and rinsed with distilled water. Mount Quick aqueous mountant (Electron Microscopy Sciences, Hatfield, PA, USA) was added to the slides, coverslipped, and sealed with nail polish. Images of the sections were captured using a ZEISS AXIO A1 Compound Light Microscope (Zeiss, Toronto, ON, Canada) with a SeBaCam 5.1MP Camera (Laxco, Bothell, WA, USA).

Whole zebrafish heads were fixed in 4% paraformaldehyde for 48 h prior to paraffin processing. Samples were transferred into 50% ethanol for 1–3 h and then placed into a cassette and loaded into a Leica Tissue Processor 1020. The processing program steps were 1 h in 70% ethanol, 1 h in 90% ethanol, 1.5 h in 100% ethanol (twice), 1.25 h in equal parts of ethanol and toluene, 0.5 h in toluene (twice), and 2 h in wax. Tissue was embedded in paraffin blocks, sectioned with a microtome, placed on slides, and dried in a 37 °C oven.
For staining, slides were treated with toluene for 10 min to remove paraffin wax and then rehydrated using an ethanol gradient (100%, 90%, 70%, and 50% ethanol for 2 min each) and washed with distilled water. The slides were stained with Hematoxylin Gill III for 2 min, rinsed with distilled water, washed with cold tap water for 15 min, and washed with 70% ethanol for 2 min. The slides were treated with eosin for 30 s and washed twice in 100% ethanol for 2 min each, followed by two toluene washes for 2 min each. The slides were covered with Dibutyl Phthalate Xylene and a coverslip and then kept at 37 °C overnight for medium solidification. Hematoxylin and eosin (H&E)-stained sections were imaged using a ZEISS AXIO A1 Compound Light Microscope (Zeiss, Toronto, ON, Canada) with a SeBaCam 5.1MP Camera (Laxco Inc., Bothell, WA, USA).