Intubation of an adult fish on an inverted microscope. (A-D) Photographs of long-term imaging of an intubated adult zebrafish inside a custom 3D-printed chamber. (A) Overview photograph of an intubation rig set up on an inverted microscope. (B) Photograph of the intubation/imaging chamber set up on a microscope stage, showing inflow tube, dual outflow tubes, overflow tube, water sensor and sponge holding fish in place. (C) Close-up photograph of the chamber with an intubated fish, with inflow tube, dual outflow tubes and water sensor noted. (D) Higher magnification image of the intubated fish. (E,F) 3D renderings of intubation chambers designed to fit on (E) a Tokai Hit heated stage (model INUB-TIZB) or (F) in a 96-well plate holder on a Tokei Hit heated stage (model STZF-TIZWX-SET), or any other stage designed to hold 96-well plates.

Schematic diagrams of zebrafish intubation. (A) Schematic diagram showing attachment of a chambered coverglass into the 3D printed chamber, using silicone grease to provide a watertight seal. (B) Schematic diagram showing a top view of the intubation chamber. (C) Schematic diagram showing a side view of the intubation chamber. (D) Schematic diagram providing an overview of the arrangement and interconnection of components for fish intubation.

Long-term time-lapse imaging of neutrophil recruitment to a scale-removal wound in an intubated adult zebrafish. (A) Schematic diagram of an adult casper Tg(lyz:DsRed2)^NZ50;Tg(mrc1a:eGFP)^y251 double transgenic zebrafish. The approximate site of scale removal by abrasion with a scalpel is noted with a yellow box. (B) An overview image of the wound area (yellow box in A) at the start of time-lapse imaging. Fluorescent neutrophils (magenta), lymphatic vessels (green) and auto-fluorescent scales (blue) are visible. The yellow dashed line indicates the boundary of the site where auto-fluorescent blue scales were removed. (C-N) Maximum intensity projection still images from long-term time-lapse confocal imaging of the adult fish in B. (C-F) Overview confocal images of the trunk at 0 (C), 3 (D), 6 (E) and 18 (F) hours. (G-J) Close-up images of the boxed regions in C-F. (K,L) High-magnification images of neutrophils (magenta) actively migrating in and around lymphatic vessels (green) in the recovering wound site of a live adult zebrafish after 23:00 (K), 23:02 (L), 23:06 (M) and 23:18 (N) of time-lapse imaging (h:min). See Movie 1 for the full time-lapse sequences, including the images in C-N. Arrows indicate two neutrophils migrating inside a lymphatic vessel. Scale bars: 1 mm in B-F; 250 µm in G-J; 25 µm in K-N.

Repeated intubation and imaging of a small wound. (A) Schematic diagram of an adult casper Tg(lyz:DsRed2)^NZ50;Tg(mrc1a:eGFP)^y251 double transgenic zebrafish. The approximate site of scale removal by abrasion with a scalpel is noted with a yellow box. (B) An overview image of the intubated fish 7 days post-wounding. Fluorescent neutrophils (magenta), lymphatic vessels (green) and auto-fluorescent scales (blue) are visible. (C-N) Confocal images taken at the beginning (0:00), halfway through (1:45) and at the end of a 3.5 h intubation that was repeated four times (on day 0, 1, 2 and 7). Outline of the wound area is indicated by a dotted line in C. Scale bar: 2.5 mm in B; 500 µm in C. See Movie 2 for full time-lapse sequences.