(A) Cartoon of 15 days of post fertilization (dpf) larval zebrafish showing the general location (green box) of images in panels B–D. (B, C) Examples of Plin-positive adipocyte lipid droplets. Fish heterozygous for the noted transgene were fed Gemma for 10 days and then stained with either LipidTOX Red (B) or Green (C) dyes for a minimum of 2 hr prior to imaging. Scale = 10 µm; standard length of all fish imaged was 5.14 ± 0.26 mm (mean ± SD, n = 47 fish). (D) EGFP-Plin2 and Plin3-RFP co-label the surface of adipocyte lipid droplets. Fish are heterozygous for each transgene and were fed Gemma + 4% cholesterol for 10 days prior to imaging. Scale = 10 µm; standard length of similarly fed double-heterozygous fish at 15 dpf, 5.19 ± 0.42 mm (mean ± SD, n = 14 fish). For B–D, images are representative of at least 10 fish per genotype from three independent clutches. (E) Darkfield whole-mount image of a 21 dpf zebrafish, yellow box indicates region of images in panels F–H; scale = 1 mm. (F) Images of pancreatic visceral adipose tissue/abdominal visceral adipose tissue (PVAT/AVAT) adipose depots in wild type and Fus(EGFP-plin2)/+; Fus(plin3-RFP)/+ fish at 21 dpf. Adipocyte lipid droplets are co-labeled with Plin2 and Plin3. Note the substantial autofluorescence in the EGFP channel in wild-type fish. (G, H) Examples of PVAT/AVAT in either Fus(plin3-RFP)/+ fish stained with LipidTOX Green (G) or Fus(EGFP-plin2)/+ stained with LipidTOX Red (H) to more clearly show the relationship of the Plin fluorescence relative to the lipid content of the droplets. For (F–H), fish were fed standard Gemma diet for 15 days and fasted for 24 hr prior to imaging to decrease the fluorescence in the intestine resulting from both EGFP-Plin2 expression and/or LipidTOX-labeled lipid droplets in enterocytes. Scale = 500 µm; standard length of the fish shown is noted in upper left corner of the darkfield images. Images are representative of 18–27 fish from two independent clutches.
