Transmitted light microscope

To measure ciliary beating in the adult zebrafish brain, we used an optimized protocol described earlier (D’Gama et al., 2021 (link), Jeong et al., 2022 (link)). We dissected the brain of nacre (mitfa^b692) adult zebrafish (male and female, less than 1 year-old) in cold artificial cerebrospinal fluid (aCSF). Then we placed the brain explant on a FluoroDish and perfused the brain explants with oxygenated artificial cerebrospinal fluid (aCSF) at room temperature. Ciliary beating of the tela choroidae in the brain explant was recorded using an Olympus transmitted light microscope, a 40 x water immersion objective (Olympus, NA 0.8) and a manta camera (Prosilica GT1930, Allied Vision) at circa 100 Hz with a resolution of 0.314 µm/pixel. Further analyses were performed in MATLAB (Mathworks).

All samples were washed, dehydrated with increasing concentration alcohol solutions (Sigma-Aldrich, St. Louis, MO, USA), and then infiltrated into methacrylic resin (Sigma-Aldrich, St. Louis, MO, USA) for the histological analysis. After, the sample was processed to obtain non-decalcified sections using a disk abrasion system (LS2—Remet, Remet, Bologna, Italy) and diamond disk cutting system (Micromet—Remet, Bologna, Italy) to obtain sample slides about 200 microns thick. Then, all samples were treated with low abrasive paper on the lapping machine (Bueheler, Lake Blu, IL, USA) with thickness control that allows for progressively reducing the sample thickness up to about 40–50 microns. Finally, the specimens were polished, colored with basic fuchsin and blue toluidine and observed with light and polarized light microscopy (Olympus, Shinjuku, Tokyo, Japan). The histological images obtained from the transmitted light microscope (Olympus, Shinjuku, Tokyo, Japan) were digitized through a digital camera and analyzed by means of an image analysis software IAS 2000 (QEA, Billerica, MA, USA). For each sample, percentage of residual bone volume with exclusion of medullary tissues (BV%), = percentage of the remaining graft, excluding bone and marrow (Graft%), and vital bone percentage with exclusion of the medulla and residual graft (VB%) were detected.

All samples were washed, dehydrated with increasing concentration alcohol solutions (Sigma-Aldrich, St. Louis, MO, USA), and then infiltrated into methacrylic resin (Sigma-Aldrich, St. Louis, MO, USA) for the histological analysis. Subsequently, the sample underwent processing to obtain non-decalcified sections using the LS2 disk abrasion system (Remet, Bologna, Italy) and the Micromet diamond disk cutting system (Remet, Bologna, Italy), resulting in sample slides approximately 200 microns thick. Afterwards, all samples were treated with low-abrasive paper on a lapping machine (Bueheler, Lake Bluff, IL, USA) with thickness control, gradually reducing the sample thickness to around 40–50 microns. The specimens were then polished, stained with basic fuchsin and toluidine blue, and examined using light and polarized light microscopy (Olympus, Shinjuku, Tokyo, Japan). Histological images obtained from the transmitted light microscope (Olympus, Shinjuku, Tokyo, Japan) were digitized using a digital camera and analyzed with image analysis software, IAS 2000 (QEA, Billerica, MA, USA). For each sample, the percentage of residual bone volume excluding medullary tissues (BV%), the percentage of remaining graft excluding bone and marrow (Graft%), and the percentage of vital bone excluding the medulla and residual graft (BV%) were measured and recorded (Figure 3).