Going beyond single gene function and analyzing gene regulatory networks requires multiple mutations in a single animal. In this system, we optimize the amount of data generated from a single tissue to correlate genetic mutations with morphologic and expression profiles. This is achieved by first injecting lipophilic die To visualize several discrete neuronal populations.
In situ two hybridization is performed to visualize the spatial temporal pattern of gene transcripts. While immunohistochemistry reflects specific patterns of protein expression, the added dimension of histological sectioning contributes to phenotype characterization.Combined. This experimental system affords the ability to analyze several different histologically recognizable structures, as well as gene and protein expression, all from the same specimen in both hole mounted and sectioned preparations Weise a system that combines lipophilic dye tracing and C two hybridization, immunohistochemistry, and histology.
This technique maximizes information gained from a single specimen while correlating morphology with gene and protein expression For all tissue dissections and manipulations. Fix the animals in 4%paraldehyde in 0.1 molar phosphate buffer by trans cardio perfusion using peristaltic pumps with appropriately sized needles. Since the DY are photo sensitive, store them in a dark closed compartment and to avoid cross-contamination, designate a separate set of instruments to handle each dye under a dissecting microscope.
Determine a dye application site to selectively label the neuronal population under consideration and not other extraneous structures. Extract extraneous tissue in order to visually confirm the chosen site and access for placement of the dye. Now cut the preloaded neuro view dye filter strips into appropriately sized triangular pieces with micro scissors.
Be sure to cut as small of a piece as possible to avoid labeling other structures for insertion into soft tissues, such as brain directly. Insert the filter using a point of the filter triangle to pierce the tissue. For more rigid tissues, make an incision to allow easier insertion of the D.Do not use a dissecting needle to push the filter into the tissue.
Place the specimens in a securely closed vial with 4%para formaldehyde and incubate at 36 degrees Celsius in the dark for two to 14 days, depending on age and diffusion distance to be covered. Using a dissecting scope with epi fluorescence, verify that the dye has diffused to the desired location and dissect out the tissue of interest. Hold mount the specimen onto a slide with glycerol for imaging with a confocal microscope.
After imaging lipophilic eyes on the confocal transfer samples to a two milliliter RNAs free eph tube with RNAs free PBS to rinse off the glycerol systematically. Process the samples through dehydration and rehydration with a graded methanol series at four degrees Celsius. Start with an incubation in 100%methanol for at least an hour and work down to 25%methanol for 15 minutes.
Wash three times in one times PBS for five minutes.Each. Digest the tissue with two microliters of 20 milligrams per milliliter Stock proteinase K in two milliliters of fresh PBS. Monitor the digestion reaction by the change in tissue opacity from opaque to almost clear.
In order to stop the enzyme digestion, incubate the samples in 4%para formaldehyde for five minutes. Then wash with PBS once for one minute, followed by three five minute washes. Discard the final PBS wash paying special attention to eliminate as much as possible as a pre hybridization step.
Incubate samples in 1.8 milliliters of hybridization. Mix for one hour at 60 degrees Celsius on an inverter denature salmon sperm DNA by incubation at 85 degrees Celsius for 10 minutes and set on ice, add 200 microliters of denatured, single stranded DNA and approximately 100 nanograms of DIG labeled riba. Probe to each sample and hybridize overnight at 60 degrees Celsius in a hybridization oven.
Process the samples with three 10 minute washes of two times SSC at 60 degrees Celsius in the hybridization oven. Importantly, remove any endogenous alkaline phosphatase activity with a wash of two times SSC for 60 minutes at 70 degrees Celsius in an ISO temp heat block. Wash the samples with PBS for five minutes.
Now replace the PBS and add one microliter of 10 milligrams per milliliter. RNAs A and incubate for 60 minutes at 37 degrees Celsius in an ISO temp heat block. Discard the PBS RNAs A and wash three times in wash solution for 10 minutes each.
Finally, heat and activate any remaining RNAs a by incubation for 60 minutes at 70 degrees Celsius in an ISO temp heat block. The final step is to visualize the ribo probe. Incubate samples in one times block buffer for one hour.
Discard the block buffer and add two milliliters of block buffer containing one microliter of anti ditch antibody to each sample. Incubate overnight wash several times with one times wash buffer as indicated. Then incubate the samples in one times wash buffer overnight.
Rinse the samples with one times detection buffer for 10 minutes and transfer the samples to a well plate. Discard the buffer and develop color with BM purple since BM.Purple is light sensitive. Cover with foil or a box when the desired signal strength is obtained.
Rinse the samples with one times detection buffer in the wells for five minutes. Image the samples with transmission microscopy or store them in 4%Para formaldehyde at four degrees Celsius. Rid the tissue of lipophilic dyes with a graded ethanol series if needed.
Then rehydrate the tissue by incrementally adding PBS over five to 10 minutes. Now block the tissue for one hour in 2.5%normal goat serum and 0.5%Triton X 100 at room temperature on a shaker incubate with primary antibody diluted in block solution for 48 hours at four degrees Celsius on a shaker wash three times with PBS for one hour each H block the samples again, then incubate with fluorescent conjugated secondary antibody diluted in block solution for 12 to 24 hours at four degrees Celsius on a shaker. From this point forward, care should be taken to block light from the sample as the secondary antibodies light sensitive.
Again, wash the samples three times with PBS for one hour each. Finally analyze the specimens by imaging with epi fluorescent and confocal microscopy. Epoxy embedding and sectioning requires fixation of tissue in 2.5%GLUTARALDEHYDE 1%PFA in 0.1 molar phosphate buffer usually overnight in a glass sample vial dehydrate the tissue with graded ethanol incubations now to transition to solvents incubating one to one absolu ethanol to propylene oxide for five minutes, followed by five 10 minute washes in propylene oxide only infiltrate the sample with resin with a simple incubation in one to one propylene oxide to resin overnight on a shaker.
Pour the solution with the sample into a flat embedding mold and leave on the counter for four to six hours to evaporate the propylene oxide. Transfer the samples to 100%resin for four hours in the final mold. Embed the samples in resin and place a label.
Allow the resin to polymerize by incubating at 60 degrees Celsius for 24 to 48 hours. In order to minimize extraneous resin, carve the resin block with a razor blade as needed. On an ultra microtome.
Cut one to two micron serial sections mount and image the samples using transmission and epi fluorescent microscopy. Different wavelength, lipophilic dyes can facilitate the visualization of central projections of three different cranial nerves. Here lipophilic die is placed into the peripheral portions of the trigeminal glossopharyngeal and vagus nerves.
After incubation for dye diffusion, the nerve central processes to the brainstem are visualized by brightfield microscopy. When the hind brain is dissected and flat mounted, some labeling of the central processes of the three labeled nerves are clearly evident. Under brightfield microscopy, the confocal microscope allows dimensionality where specific are illuminated, and the use of three different dyes allows for the assessment of the distribution of each population.
In relation to the others, here, the inner ear afferent are traced in a newborn mouse by neuro view. Red in C two hybridization for prox one, and a combination of immunoreactivity to anti tubulin and anti prox. One to visualize the afferent.
In addition, a two micron thick epoxy resin section images the cochlear as a cross section action. When attempting this procedure, it is important to carefully choose the placement of lipophilic eye. A good understanding of neuroanatomy will help in this process also be cautious of all photos sensitive steps.
This procedure will reduce the amount of breeding and this time needed to obtain publishable data while improving information about colocalization and cove effects in mutants.
