TITLE: Protocol (B): Zebrafish embedding and imaging (3 dpf)
AUTHORS: Désirée A. Schmitz, Tobias Wechsler, Hongwei Bran Li, Bjoern H. Menze, Rolf Kümmerli

[DESCRIPTION]
This protocol details the zebrafish embedding and imaging.

[STEPS]
SECTION: Part 0: Material preparation
1. Prepare 1.5% low-melting point agarose by heating e.g. 1.5 g low-melting point agarose (Sigma, serial no: A9414) in 100 mL distilled water in a flask (minimal volume 150 mL) in 30 s bursts in the microwave. 
SECTION: Part 0: Material preparation
2. Once the solution is clear, i.e. no flocs or powder are visible anymore, aliquot into 1.5 mL Eppendorf tubes. 
SECTION: Part 0: Material preparation
3. The day before embedding, prepare an 30 s culture of all bacteria used for injections. These will serve as a positive control. 
SECTION: Part 1: Embedding zebrafish in low-melting point agarose
4. Heat 1.5% low-melting point agarose in 1.5 mL Eppendorf tube aliquots to 90 °C in a heating block.  
SECTION: Part 1: Embedding zebrafish in low-melting point agarose
5. Once the agarose has melted, reduce the heat to 42 °C. 
SECTION: Part 1: Embedding zebrafish in low-melting point agarose
6. Check fluorescence in the otic vesicle of individual zebrafish using any microscope with sufficient fluorescence sensitivity and magnification. This step is only needed if the goal is to solely image zebrafish with an ongoing infection. These fishes can then be picked selectively based on their fluorescent signal in the otic vesicle. 
SECTION: Part 1: Embedding zebrafish in low-melting point agarose
7. Prepare a sufficiently large volume of anesthetic solution to use for all treatments: For this, add approximately 9 mL of E3 zebrafish water with PTU (final PTU concentration: 0.003%) and 10 drops of the thawed anesthetic tricaine (ethyl 3aminobenzoate methanesulfonate salt analytical standard, 4000 mg/L) using a Pasteur pipette (LLG Labware, 3 mL, unsterile) into a small petri dish (Greiner, 60x15mm, sterile).  

SECTION: Part 1: Embedding zebrafish in low-melting point agarose
8. For each treatment, move a few drops of the solution from the previous step into a separate small petri dish. This allows anesthetizing each treatment group individually to avoid cross-contamination. 
SECTION: Part 1: Embedding zebrafish in low-melting point agarose
9. Move the zebrafish of the first treatment into the anesthetic liquid in a designated petri dish (Fig. SP3A). 
 


 

SECTION: Part 1: Embedding zebrafish in low-melting point agarose
10. Add 4-5 zebrafish of one treatment into a well of an 8-well Ibidi µ slide (Vitaris, serial no: 80827-IBI) (Fig. SP3B).

 

 

SECTION: Part 1: Embedding zebrafish in low-melting point agarose
11. Using a Pasteur pipette, add about 5 drops of the low-melting point agarose (from the 42 °C in the heating block) to the zebrafish in a well. 

 

SECTION: Part 1: Embedding zebrafish in low-melting point agarose
12. Using a dissecting needle, mix the agarose and the small amount of liquid that was transferred with the zebrafish in the well so that the agarose concentration is the same throughout. 

 

SECTION: Part 1: Embedding zebrafish in low-melting point agarose
13. Position the zebrafish with the dissecting needle so that the injected otic vesicle (see protocol A) is on the bottom of the slide to enable the use of an inverted widefield microscope for imaging (Fig. SP3C). Do this step with the naked eye as well as checking under a stereomicroscope. 
 


 

SECTION: Part 1: Embedding zebrafish in low-melting point agarose
14. Wait approximately 5 min until the agarose has completely hardened. 

SECTION: Part 1: Embedding zebrafish in low-melting point agarose
15. Add E3 zebrafish water very carefully on top to ensure a constant supply of moisture. 
SECTION: Part 1: Embedding zebrafish in low-melting point agarose
16. Repeat steps 9-15 for all zebrafish that need to be embedded for imaging. 
SECTION: Part 1: Embedding zebrafish in low-melting point agarose
17. Add a positive control for each tagged bacterial species/strain, i.e., ~10 µL 5 min culture mixed with agarose into one well per species. 
SECTION: Part 2: Imaging the inner ear structure (otic vesicle) of zebrafish with a widefield microscope
18. Place up to 4 Ibidi slides into the slide holder in a widefield microscope. 
SECTION: Part 2: Imaging the inner ear structure (otic vesicle) of zebrafish with a widefield microscope
19. Set up the brightfield and all required fluorescence channels, e.g., GFP (excitation at 475 nm & emission 520 nm), and mCherry (excitation at 555 nm & emission at 605 nm).  
SECTION: Part 2: Imaging the inner ear structure (otic vesicle) of zebrafish with a widefield microscope
20. Set the positions of all otic vesicles with a 20X objective that has a long working distance (minimum 1 mm). 
SECTION: Part 2: Imaging the inner ear structure (otic vesicle) of zebrafish with a widefield microscope
21. Image all saved positions of zebrafish otic vesicles from the previous step within the brightfield and fluorescence channels. 
SECTION: Part 2: Imaging the inner ear structure (otic vesicle) of zebrafish with a widefield microscope
22. Save the images as .tiff files for further processing, also keeping the original images from the imaging software.