This experiment describes the process of inserting zebrafish larvae with low percentage low melting agarose for time-lapse microscopy. Depending on the scientific question, different stages of development can be observed in agarose-embedded larvae: this can include the first 48 h of development within the egg shell, or the time after hatching (48-120 h after fertilization).
1. Relevant documents
• Instruction manual for μ-Slide 2 Well Co-Culture
2. Materials and reagents
2.1. Animals
• Zebrafish larvae (up to 120 hours after fertilization)
2. Buffers and solutions
Fish water (60 times)
• 172 g of sodium chloride (ROTH, P029.2)
• 7.6 g KCl (ROTH, 6781.1)
• 29 g CaCl2 x 2 H2O (ROTH, CN92.2)
• 49 g MgSO4 x 7 H2O (ROTH, P027.1)
• Add detached water up to 10 liters
Fish water (1x)
• 160 ml fish water (60 times)
• 20 ml of 0.01% (w/v) methylene blue (Sigma-Aldrich, M9140) in deionized water
• Add detached water up to 10 liters
Tricaine stock solution
• 4 g / l Tricaine / MS-222（Sigma Aldrich，A5040）
• 0.02 M chloroform (ROTH, 4855.2), pH 9
• In deionized water
Tricaine uses a solution
• Dilute Tricaine stock solution in fish water (1x) to a final concentration of 160 mg/L (1:25)
0.5% Low Melting Agar (LMPA)
• Can be prepared in batches and divided into small portions for each experiment
• Boil 0.5% (w/v) low melting agar (Biozym, 840101) with fish water (1x) in a microwave oven until the agar is completely dissolved
• Allow the agar to cool to about 35 °C
• Add Tricaine stock solution at a final concentration of 80 mg/L (1:50)
The final low melting agar concentration can be adjusted. For example, we successfully used up to 2% low melting point agar, but decided to experiment with 0.5% low melting agar because it is unlikely to affect physiological processes.
2.3. Equipment
(ibidi, 81806)
• Thermomixers (e.g., Eppendorf, ThermoMixer C)
• μ-Slide 2-well co-culture slides, ibiTreat bottom treatment (ibidi, 81806)
• Plastic pipettes
• Optional: Small brush
•microscope
3. Experimental steps
Preparations
Before starting the experiment, prepare the LMPA as described in the "Buffers and solutions" section. Keep at 35 °C until needed. If the agar has been prepared in advance, it can be reheated in a hot mixer (~80°C) and then allowed to cool the agar (~35°C).
loading
Read the instructions before using μ-slide 2-well co-culture slides.
In some of our group's experiments, mechanical injuries were made to the spinal cord in zebrafish. If this is done, it is recommended to allow the juveniles to recover for at least 30 min before loading. After spinal cord cross-section, the initial survival rate of embedded agar is 80%. The juveniles were also alive after 48 hours of observation in agar. The initial survival rate of uninjured juveniles is close to 100%.
Anesthetize zebrafish juveniles using chloroform solution for 2 min.
Place one juvenile fish in each well of the μ-slide 2-well co-culture slide using a plastic pipette and use some excess fish water.
Perform the following steps individually for each juvenile (to avoid drying out):
3. Use a plastic pipette to remove all fish water.
3. Add 50 μl LMPA to the wells to cover the juveniles.
3. Use a brush or small pipette tip to position the juvenile in the appropriate position (lateral position, as horizontal and upright as possible, with uniform alignment, i.e. always to the left with the yolk at the bottom).
Allow the LMPA gel to solidify (about 20-30 min for 0.5% gel).
Optional: Cover the solidified LMPA gel with 1x fish water containing 80 mg/L chloroform stock solution for anesthesia for continuous imaging.
6. Cover the μ-slide 2-well co-culture slides with the included lid to avoid evaporation.
7. Zebrafish juveniles are loaded and ready for imaging
During the 48 h imaging period, if the lid is tightly closed, there is no need to add fish water above the agar as the specimen will not dry out. This also makes it possible to observe upright microscopy without fear of spillage.
Figure 1: Zebrafish larvae embedded in an LMPA μ-Slide 2-well co-incubator.
Time-lapse microscopic observation
This method of sample preparation is used for continuous time-lapse microscopy, as well as for taking snapshots of certain fields of view at different time points using various microscopes at the optical microscope facility, the core facility of the Technical University of Dresden's CRTD. This includes inverted and upright, wide-angle and confocal microscopes.
Figure 2: A) Brightfield tile scan of the entire μ-slide 2-well co-culture after burying agar 72 h after fertilization of zebrafish larvae, for a total of 18 juveniles.
Video: This example shows the Z-projection of a short 20-min three-dimensional timeline showing the transgenic mpeg1:mCherry zebrafish larvae treated with mechanical spinal cord stomy 3 days after fertilization. The fluorescently labeled cells are macrophages and microglia. This timeline was acquired using the Dragonfly Rotating Disk microscope (Andor). See the video below: