This three dimensional slide preparation method preserves the 3D chromatin arrangement of cells as demonstrated with testicular germ cells of mice. First, perme the ferous tubules to remove the cytoplasmic materials and improve the accessibility of staining reagents such as antibodies and fish probes. After fixing the nuclear materials, mechanically dissociate the germ cells with forceps and cytosine the samples onto slides.
Next, acquire data with multiple Z sections to cover a nucleus in 3D when applied strategically results from 3D slide preparations. Using COT one RNA fish can show nuclear nascent RNAs to detect the transcriptionally active regions in the nucleus. I developed this method when I was in the gene laboratory at Massachusetts General Hospital.
At that time, I optimized the slide preparation method for cotton RNA fluorescent insight to hybridization, also called fish to detect nacent.RNA. Cotton probes can hybridize repetitive sequences in introns and tls and visualize the site of active transcription. Cotton RA fish require preservation of nuclear RNA distribution.
The key point of this right preparation method is that the ization steps need to proceed The fixation step. To remove Cy Plasmic background, Unravel the OUS tubules from a ruptured testis in PBS on ice transfer several pieces of tubules of five to 10 millimeters in length to 500 microliters of CSK buffer with 0.5%tritton X 100, and incubate for six minutes on ice. Now transfer all the tubules to 4%P-F-A-P-B-S solution in one well of a four well dish incubate for 10 minutes at room temperature.
Next, incubate all the tubules in PBS for five minutes at room temperature. Set up the cyto spin and cyto spin chambers during the incubation periods on the backside of a glass slide, transfer all the tubules into 30 microliters of PBS. Then use the tips of two forceps to tear the tubules to pieces.
Chop the tubules in a horizontal direction by clipping the tubules between the tips of two forceps and pulling the forceps horizontally. After adding 30 microliters of PBS, mix the suspension by pipetting, then transfer it to a micro centrifuge tube and add PBS to a total volume of 1.3 milliliters. Now apply 100 microliters of the suspension to each of the cyto spin chambers.Cyto.
Spin the samples at 2000 RPM for 10 minutes at room temperature. Dry the slides on a lab bench for a few minutes at room temperature using a coplan jar containing PBS. Wash the slides for five minutes at room temperature.
Then transfer the slides to 70%ethanol for at least two minutes. In a 20 microliter reaction add 100 nanograms caught, 1D NA probe, 20 millimolar ribonucleotide vanadyl complex and hybridization buffer. Using a PCR machine, incubate for 10 minutes at 80 degrees Celsius, followed by prea.
Kneeling step at 42 degrees Celsius until ready for use. Now successively dehydrate the slides in 80%ethanol for two minutes, followed by 100%ethanol for two minutes. Dry slides completely on lab bench at room temperature.
Now place the slide on a tip box chamber at 42 degrees Celsius. Next, briefly centrifuge the preneed probes and pipette them directly onto the dehydrated slides. Gently place a cover slip over the slide for the hybridization step of at least six hours at 42 degrees Celsius.
Preheat the wash solutions in coplan jars at 42 degrees Celsius for 30 minutes in a water bath using a razor blade, remove the cover slip from the edge of the slide without scratching the samples progressively immerse the slides in the wash solution for five minutes each. Starting with the first coplan jar containing two XSSC and 50%form amid, followed by the second coplan jar containing two XSSC and 50%form amid. And then the last two jars containing two XSSC.
Then add 20 microliters of DPI mounting media to the samples and place a cover slip. Gently press the cover glass and blot the extra mounting media with a cleaning tissue. In this experiment caught one RNA fish was performed to simultaneously detect nascent transcription and immunostaining of proteins that localize to the XY body immunohistochemistry.
On this PACU, spermatocyte detected the heterochromatin protein CCB X one and also a phosphorylated form of histone variant H two A X on the XY body. The resulting fish caught one signals accumulated on the e chromatic regions in surface spreads treated with hypotonic solution. The three-dimensional chromatin structure is disrupted and all myotic chromosome axes can be captured within a single Z plane.
3D slide preparation, however, preserves the intact chromatin structure Following this procedure. This right preparation method is applicable for immunofluorescence DNA and RNA fish, and the combination of these detection methods.
