FIGURE SUMMARY
Title

Beyond the whole-mount phenotype: high-resolution imaging in fluorescence-based applications on zebrafish

Authors
Oralová, V., Rosa, J.T., Soenens, M., Bek, J.W., Willaert, A., Witten, P.E., Huysseune, A.
Source
Full text @ Biol. Open

Imaging of transgenic zebrafish lines. (A,D,G) Whole-mount, (B,E,H) low- and (C,F,I) high-magnification (B,E,F,H,I) cross sections and (C) sagittal section. Lines in A,D,G indicate approximate level of sectioning in B,E,H and C,F,I. (A–C) Tg(sox17:egfp) zebrafish with GFP-positive endodermal cells along the midline, extending into the pouches (arrows). Blood vessels also show a fluorescent signal. (D–F) Tg(krt4:gfp) zebrafish with GFP-positive periderm (arrows). Labeled cells also cover the oropharyngeal lining (arrowheads). (G–I) Et(Gal4-VP16)zc1044A;Tg(UAS-E1b:nsfB-mCherry)c264, abbreviated as GET-periderm line (GET, Gal4 enhancer trap) (Eisenhoffer et al., 2017). The periderm is labeled red (arrows). b, brain; e, eye; nt, notochord; ov, otic vesicle; y, yolk. Scale bars: (A,D) 250 µm; (E,G,H) 100 µm; (B,C,F,I) 50 µm.

Imaging of photoactivatable kaede. Vertebral end plates of an adult Tg(osx:kaede) zebrafish shown using a GFP (A–C) and rhodamine (A′–C′) filter, before (A,A′), after 5 s (B,B′) and 30 s (C,C′) of exposure to a wavelength of 365 nm. Note the fading of the green signal from A to C and strengthening of the red signal from A′ to C′ in the osteoblasts lining the vertebral end plates (arrowheads). All pictures were taken using the same exposure time (1.6 s). Scale bar: 25 µm.

Whole-mount immunostaining. (A,D,G) Whole-mount, (B,E,H) low- and (C,F,I) high-magnification cross sections. Lines in A,D,G indicate approximate level of sectioning in B,E,H and C,F,I. (A–C) Wild-type (WT) zebrafish immunostained for laminin. Note the basal lamina delimiting optic cup, brain, epidermis and endodermal pouches. (D–F) Tg(sox17:egfp) zebrafish pulse-labeled with BrdU and whole-mount stained with an anti-BrdU antibody. (G-I) Tg(sox17:egfp) zebrafish immunostained with a pan-cytokeratin antibody. The whole-mount images in D,G show the red channel only; the GFP fluorescence is likewise visible prior to dehydration and embedding. b, brain; e, eye; ep, epidermis; oc, optic cup; p, endodermal pouch. Scale bars: (A,D,G) 250 µm; (E,H) 100 µm; (B,F,I) 50 µm; (C) 25 µm.

Cell tracking with fluorescent vital dyes and on-section histochemistry. (A–I) Cell tracking. (A,D) Whole-mount, (B,E) low- and (C,F) high-magnification cross sections. (A–C) WT zebrafish injected with DiI (A, arrow) at 28 hpf and euthanized after 48 h reveal areas of DiI distribution (arrowheads); the DiI label (yellowish dots) stands out sharply against an autofluorescent background (B,C). (D–F) WT zebrafish vital stained with CDCFDA. Only cells exposed to the solution (periderm) take up the stain (E, arrows). After a chase time of 22 h, labeled cells can be observed inside the forming gill slit (F, arrowhead). Lines in D indicate approximate level of sectioning in E,F. (G–I) DAPI staining (G) on section of CDCFDA labeled embryo (H) and merged picture (I). (J–K) On-section histochemistry for the osteoclast marker TRAP, showing the palatoquadrate and palatine bone in adult WT zebrafish, viewed in transmitted light (J), under epifluorescence (K) and the overlay of transmitted light and epifluorecence (L). b, brain; e, eye; m, mouth; nt, notochord; ph, pharyngeal lumen. Scale bars: (A,G,H,I) 100 µm; (D) 250 µm; (B,E,F) 50 µm; (C,J,K,L) 25 µm.

 
 

Acknowledgments
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