The overall goal of this protocol is to isolate neurons, macrophages, and microglia from larval zebrafish brains and to extract high-quality RNA to perform downstream analysis such as qPCR and transcriptomics. Transgenic larval zebrafish are a powerful tool for live imaging and provide us with the opportunity to observe individual cells like microglia in their living environment over time. However, to gain a detailed understanding of their functions we need to understand their gene expression profile, and our isolation and sorting protocol is designed for this purpose.
The main advantage of this technique is that it can isolate different types of cells from the central nervous system with minimal modification to their gene expression profile, so that cell functions and properties can be characterized. The efficiency of this protocol is reflected in its efficacy. With this protocol, sufficient amounts of RNA can be produced within short periods of time for various downstream applications.
After raising zebrafish embryos in E3 medium containing PTU according to the text protocol, use a fluorescent stereomicroscope to screen larvae at two dpf for GFP-positive macrophages and microglia in DsRED-positive neurons. To homogenize the embryos at one point five ml of 15 millimolar Tricaine per 50 ml of medium to 50 larvae to prepare anesthesia. Then use a three ml Pasteur pipette to transfer 10 larvae at a time into a 55 ml Petri dish filled with ice-cold E3 medium with Tricaine to terminally anesthetize them.
Under a steromicroscope, align 10 larvae in the center of the Petri dish, then using surgical micro-scissors transect the larval heads above the yolk sac. With a three ml Pasteur pipette, take up all the heads and with as little liquid as possible, transfer them into a glass homogenizer on ice containing one ml of ice-cold Media A.Replace each small Petri dish containing ice-cold E3 plus Tricaine with a new one every 30 minutes to assure that transection is performed in cold E3 plus Tricaine medium. Replace the ice-cold Media A in the glass homogenizer when the color starts fading.
Once the entire group of heads has been collected, remove all the Media A from the glass homogenizer and replace it with one ml of fresh ice-cold Media A.With the homogenizer still on ice, use a tight-fitting pestle to disrupt the brain tissue by performing 40 rounds of crushing and turns for three to five dpf larvae and 50 rounds for seven and eight dpf larvae. Then add two ml of Media A to the cell suspension to dilute the cells and reduce their agglomeration with myelin. Eliminate cell agglomeration by running the cell suspension through a 40 micron cell strainer into a cold 50 ml falcon tube on ice.
Repeat this step three times. Transfer one ml aliquots of cell suspension into cold one point five ml tubes, and spin them at 300 times g and four degrees Celsius for 10 minutes. Then, using a 10 ml syringe with a 23-gauge one inch needle remove the supernatant.
With one ml of ice-cold 22 percent density gradient medium gently overlaid by zero point five ml of ice-cold 1X dbps, gently re-suspend the cell pellet. Spin the tubes at 950 times g without a break in slow acceleration at four degrees Celsius for 30 minutes. After the spin, discard the dbps density gradient medium in myelin trapped at their interphase.
Then use zero point five ml of Media A with two percent NGS to wash the cells, and spin the tubes at 300 times g at four degrees Celsius for 10 minutes. Remove as much supernatant as possible, then pull the cell pellets from the same experimental condition together in one ml of Media A with two percent NGS. If the cells of interest express a fluorescent protein run the cell suspension through a 35 micron cell strainer cap and transfer them into cold 5 ml FACS tubes on ice protected from light.
To immunostain microglia, use zero point three ml of Medium A plus two percent NGS to re-suspend the cell pellet, then split the cells between three one point five ml tubes, one for unstained cells to measure autofluorescence, one to measure the nonspecific binding of a secondary antibody to microglia and a third as a test. To all the tubes, add one percent low endotoxin azide-free or a leaf to block CD16, CD32 interactions with the FC domain of immunoglobulins. Then incubate the cells for 10 minutes with gentle agitation every five minutes.
Next, to the third tube add the 4C4 antibody and incubate it for 30 minutes with gentle agitation every 10 minutes. Then spin the tubes at 300 times g and four degrees Celsius for 10 minutes. After discarding the supernatant, wash the pellet once with zero point five ml of Media A plus two percent NGS before spinning the tubes again.
Re-suspend the cell pellet with zero point five ml of Media A plus two percent NGS, then add one percent leaf and incubate the cells for 10 minutes with gentle agitation every five minutes. To tubes two and three, add secondary antibodies and place the tubes in the dark. After spinning the tubes and discarding the supernatant, use zero point five ml of Media A plus two percent NGS to wash the samples twice, then re-suspend the cell pellets with one ml of Medium A plus two percent NGS.
Run the cell suspension through a 35 micron cell strainer cap and transfer it into cold five ml FACS tubes on ice protected from light. Finally, carry out FACS sorting and RNA extraction according to the text protocol. In this study, neurons and macrophages plus microglia were isolated from eight dpf mpeg1 EGFP-positive NBT dsRED-positive larvae.
FACS was used to separate the cells from debris by function of their size and granularity. Single cells were then separated from doublets or cell agglomerates. From the single cell population, a gate was drawn to eliminate dead cells.
The corresponding dot plot revealed that this experimental protocol preserves cell plasma membrane integrity as the rate of dead cells is only 26.7 percent. As shown here, neurons and macrophages plus microglia were easily segregated from the live cell population gates. Within the brain, the neuron population appeared to be more prominent than the macrophage and microglia population.
In a second study, FACS sorting was used to isolate live microglia from larval brains with 4C4, an antibody that specifically labels microglia. As summarized in this table of microglia isolation and RNA extraction data, the number of microglia within zebrafish larval brains vary and are very low at three dpf. Finally, RNA extraction results obtained with one experiment from microglia of five dpf larval zebrafish brains is shown in this electrophoresis trace with a clear visualization of ribosomal RNA.
Once mastered, this technique can be done in 12 hours depending on how many heads are needed per condition. While attempting this procedure, it's important to remember to keep everything cold and the surfaces clean to avoid RNA degradation. After its development, this technique paves the way for researchers in the field of neurosciences using zebrafish as a model to explore gene expression profiles of different cells under physiological and pathological conditions.
After watching the video, you should have a good understanding of how to isolate neurons, macrophages, and microglia from larval zebrafish brains, and how to extract high-quality RNA to perform downstream analysis like qPCR and transcriptomics.
