This protocol offers a relatively rapid and cost efficient method, as well as an automated acquisition and analysis thus producing low sensitivity screening of 5-Methylcytosine which is a common epigenetic biomarker. The main advantage of this technique is in the flexibility it offers an experimental design that users can optimize to their model and stage of development. This technique can be used to detect how chemical exposure alters the relative abundance of 5-Methylcytosine within a developing organism which normally could only be done through expensive high trpA methods such as sequencing.
This method is useful to any research which aims to utilize NCG methods to understand epigenetic alterations of 5-Methylcytosine in response to environmental stimuli. Begin by preparing a fresh exposure solution on the morning of collection. Add five milliliters of particulate free system water to a 60 millimeter glass Petri dish.
Then use a 10 microliter micro capillary glass pipette to transfer 10 microliters of dimethyl sulfoxide or chemical stock solution to system water. Swirl the glass Petri dish to ensure the stock solution dissipates. Add another five milliliters of system water to the glass Petri dish and swirl the dish to homogenize the exposure solution.
Cap the glass dishes with glass lids to minimize evaporation. Next, use an RO water-filled squirt bottle to transfer the embryos from the fish net into a 100 millimeter dish. Randomly sort 50 live embryos at 0.75 hours post fertilization and remove excess RO water.
After the exposure duration has ended, remove the embryos from the incubator. Transfer all living embryos to a 1.5 milliliter micro centrifuge tube. Aspirate residual exposure solution without aspirating any embryos.
Add 500 microliters of chilled 4%paraformaldehyde and mix the embryos by inversion several times. Allow the embryos to fix and 4%paraformaldehyde overnight at four degrees celsius. After fixing the embryos aspirate the 4%paraformaldehyde, respend the embryos in 1X PBS, and mix by gentle pipetting for 30 seconds.
Using a glass pipette, carefully transfer fixed embryos to an RO water-filled glass dish. Gently swirl for 30 seconds and repeat this wash once. Under a stereo microscope set at five times magnification decorenate the embryos using syringe needles.
Using the needle tip, puncture the chorion and gently peel it away from the yolk and cell mass. After the embryos have been decorenated, use a glass micro capillary pipette to transfer up to 25 intact embryos into one immunohistochemistry basket. Place the IHC basket into a 24 well plate.
Then aspirate the water from each well. Resuspend the embryos in 500 microliters of blocking buffer per well, wrap the plate with paraform and aluminum foil to protect it from light. Incubate the plate at four degrees Celsius for four hours on an orbital shaker at 100 rpm.
After incubation, aspirate the blocking buffer from each well. Replace it with 500 microliters of primary antibody solution. Remember to incubate a subset of vehicle control embryos with the blocking buffer to account for background noise.
Rewrap the plate and paraform and aluminum foil and allow the plate to incubate at four degree Celsius overnight on an orbital shaker. The following day remove the primary antibody solution from each well and replace it with 1x PBST. Wash each well thrice with 1x PBST for five minutes per wash on an orbital shaker.
Aspirate 1x PBST from each well and place the IHC basket into a glass Petri dish filled with RO water. Under a standard stereo microscope randomly sort intact embryos into a 96 well plate ensuring one embryo per well. Then remove all RO water from individual wells and add 200 microliters of 1x PBS.
Centrifuge the plate for three minutes at one G.Image the embryos with a two times objective under transmitted light and FITC. Select autofocus to ensure the focus of the camera is on the bottom of the plate and offset by the bottom thickness. Select a FITC wavelength with an exposure duration of 100 milliseconds and set the auto focus to laser with a Z offset.
Observe and confirm proper image acquisition and several wells prior to commencing plate acquisition. After image acquisition, carry out data analysis using the high content screening system and a custom automated image analysis procedure. Select each embryo on the 96 well plate to be analyzed for total area and integrated intensity of fluorescence.
Then tabulate the data points and export them from the high content screening system by going to measure and selecting open data log to export the data to a spreadsheet. Upload the exported spreadsheet to the deployer package to sort and summate data for each well and filter by well number. Then use the right Excel package to export the summated data into a spreadsheet.
The distribution and integrated intensity of 5-Methylcytosine specific total area were assessed for Zebrafish embryos at six hours post fertilization. The x-axis shows exposure to either DMSO as a vehicle or TDCIPP as the positive control. The Y-axis denotes relative fluorescence.
The thresholds were significantly different from the vehicle treated embryos as shown by the total area of 5-Methylcytosine detected within embryos and the integrated intensity within that same area indicating maximum signal to noise ratio. It is extremely important to maintain the integrity of the yoke and cell mass for proper IHC detection. This method is relatively insensitive and only offers relative abundance RFU data.
If quantification is required one can follow up with a more targeted approach.
