The protocol described here allow us to investigate the function of a chemokine and the behavior of macrophage in vivo. Most of existing experimental models of cell chemotaxis are based on in vitro cell experiments. But in vitro experiments sometimes are too simple to model the complex environment in vivo.
Also, they may not represent the chemoattraction ability in vivo. These methods utilize the specific advantage of zebra fish for direct cell behavior observation, which is difficult for mice. Start by generating TGFABP-10A interleukin-34 transgenic constructs.
Inject the FABP-10A interleukin-34 constructs into one cell stage Tg(mpeg1:GFP)transgenic and a wild-type fish embryos together with the transposase MRNA. Raise and collect the embryos according to manuscript instructions. Then, fix them with 4%paraformaldehyde overnight at 4 degrees Celsius or for two hours at room temperature.
After fixation, wash the embryos with PBST three times for five minutes per wash. Dehydrate the embryos separately with 50%methanol in PBST, followed by 100%methanol, then change to fresh 100%methanol, and store them at negative 20 degrees Celsius for at least 2 hours. When ready for probe hybridization, rehydrate the embryos separately in 50%methanol in PBST.
Then, wash them with PBST three times for five minutes per wash. Digest the embryos with proteinase K in PBST at room temperature. Then, discard the digestion solution, and repeat fixation with 4%paraformaldehyde for 20 minutes at room temperature.
After fixation, wash the embryos twice with PBST for 10 minutes per wash. Perform prehybridization with heated hybridization buffer at 65 degrees Celsius for at least one hour. Then, preheat the interleukin-34 probe to 65 degrees Celsius for 10 minutes.
Recycle the hybridization buffer into the original tube and perform hybridization with the preheated probe at 65 degrees Celsius overnight. On the next day, wash the embryos with 50%formamide and 2X SSCT followed by 2X SSCT and then 0.2X SSCT at 65 degrees Celsius. Repeat each wash three times with 20 minutes per wash.
Then, wash the embryos three times with PBST for five minutes per wash. Block the samples with 600 milliliters of blocking buffer for one hour at room temperature. Then, add 400 microliters of anti-digoxigenin HRP antibody solution and incubate the embryos at four degrees Celsius overnight.
On the next day, remove the antibody and wash the embryos six times with PBST for 20 minutes per wash. After the last wash, rinse each sample with 30 microliters of 1X plus amplification diluent for five minutes. Incubate the samples in the fluorophore tyramine working solution for five to 15 minutes in the dark.
Extending the incubation time to 30 minutes if signals are weak. Then, wash the embryos with PBST three times for 10 minutes per wash. And incubate them with the primary antibody at four degrees Celsius overnight.
On the next day, wash the embryos five times with PBST for 30 minutes per wash, and incubate them with the secondary antibodies at four degrees Celsius overnight. On the next day, wash the embryos three times in PBST for 10 minutes per wash, and store them in 70%glycerol in the dark at four degrees Celsius or negative 20 degrees Celsius for longer. Prior to imaging, use a fluorescence microscope to select the DS red and GFP double positive embryos.
To mount the fish, use a metal bath to heat one milliliter of 1%low melting agarose to above 90 degrees Celsius. Then, cool it to body temperature and mix in 50 microliters of 0.2%tricaine. Transfer the anesthetized embryos to a small dish mounted with a cover slide on the bottom.
Remove the surrounding water and slowly drop the low melting agarose on the embryos. Carefully set the position of the fish before the agarose has solidified, keeping the liver area close to the cover slide. Once the agarose has solidified, carefully cover it with another layer of agarose to reinforce it.
Place the dish on the confocal microscope carrier table, cover the fish with the E2 solution with tricaine, and start imaging. To operate the confocal microscope, open the Zen Black 2.3 software and click locate, then incubation, and then set the temperature to 29 degrees Celsius. Click the acquisition menu and select the required scan mode and lasers in the smart setup menu.
Then select Z-stack and position. Click on the experimental designer menu and select enable multi block experiment in the first block to find the sample under low magnification. Then, switch to the high magnification and let the observed area in the center of the visual field.
Set the position and Z-stack information. Then, select the appropriate laser intensity, scanning layers, and imaging speed. Once all blocks are setup, set the appropriate number of loops and start recording.
This protocol was used to inject a liver-specific interleukin-34 over expression plasmid into transgenic fish embryos, who's macrophages were labeled with GFP. Full mount fluorescence in situ hybridization and immunostaining were used to analyze the expression of interleukin-34 and the GFP labeled macrophages. Macrophage cell numbers were quantitatively analyzed in uninjected and construct injected embryos'liver and tail region.
Live imaging was used to directly observe macrophages, labeled green, passing by the liver of a control fish and migrating into the liver of an interleukin-34 over expressing fish. The important steps of this protocol include the selection of a suitable transgenic line to label the cell of interest. And appropriate tissue for imaging your expression of transgenic gene as well as an appropriate observation time window.
This technique could be applied to study the function of other chemokines on the behavior of other cells, such as T-cells and neutrophils in vivo.
