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Fig. 1
Overview of key steps in sample preparation for optical projection tomography (OPT). (A) Summary of 8 step workflow for sample preparation for OPT scanning. (B) Intraperitoneal injection of 40 μL of EdU into ventral abdomen of adult zebrafish using a 1 mL syringe and 30 gauge × ½ inch needle. Note the use of V-shaped holder to orient and stabilize the anesthetized specimen during injection. (C) Dorsal view of adult zebrafish brain in situ prior to excision and fixation. (D) Excised adult zebrafish brain fixed in 2% paraformaldyhyde. (E) Representative image of three adult brains in EdU staining solution in a 12-well plate. (F) Adult brain embedded dorsally and centered in well and in z-plane in low melting agarose in a 6-well plate. (G) Low melting agarose cylinder removed from 6-well plate in preparation for trimming. (H) Initial trimming using a razor blade to form a trapezoid by 4 sequential cuts: (1) perpendicular to olfactory bulbs, (2) perpendicular to and ~1 cm from spinal cord, and (3 and 4) two lateral diagonal cuts joining 1 and 2 together. (I) Trapezoid oriented upright with brain positioned along the long-axis vertically. Olfactory bulbs are localized at the top of the block. (J) Trimmed block ready for dehydration and clearing. Notice block is tapered from top to bottom to reduce agarose around brain sample for scanning and to provide a larger base to adhere to mount. (K–L) Position of brain within trimmed block viewed under brightfield observed along the long-axis (K) and from the dorsal aspect of the block (L). (M) Adult zebrafish brain (black arrow) observed en block following methanol dehydration and BABB clearing. Note the transparent nature of the brain. (N) Sample adhered to an OPT mount in preparation for scanning. In all panels, the corresponding detailed protocol steps are denoted in the bottom right-hand corner.