The overall goal of this procedure is to visualize patterns of mRNA expression in developing fetal mouse lower urogenital tract tissues. This is accomplished by first synthesizing a di oxygen in labeled ribo probe from a PCR generated template. The second step of the procedure is to generate thick cut sections using a vibrating microtome.
The third step is to conduct an in situ hybridization in lower urogenital tract sections contained in customized baskets. The mRNAs are visualized through immunohistochemical detection of bound ribo probes using BM purple precipitating alkaline phosphatase substrate. Ultimately, microscopy can show the spatial and temporal localization of low and high abundance mRNA transcripts in the fetal mouse lower urogenital tract.
The main advantage of this technique over existing methods like immunohistochemistry is that ribo probes are easy to generate and can be made to recognize most mRNAs in the entire mouse genome. Though this method provides insight into mRNA expression patterns during fetal mouse genital urinary development, it can also be applied to other organ systems, species and developmental stages. Visual demonstration of this method is critical as embedding the tissue in auger can be difficult to learn, and because slight variations in orientation can lead to inconsistent results At least one day before preparing the tissue, remove and discard the membrane from a 12 millimeter diameter milli cell culture plate while insert the remaining ring will function as a mold soak the rings overnight in RNAs inhibitor solution.
On the day of the protocol, dissect a mouse lower urogenital tract that has been rehydrated with graded methanol. PBS tween washes discard approximately two thirds of the bladder and store the remaining lower urogenital tract tissue in PBS tween. Now place a treated ring mold on a glass microscope slide and fill it with 4%low melt arose solution at 62 degrees Celsius and allow it to cool for about two minutes.
Now, block dry the lower urogenital tract tissue and submerge it in the arose solution. Use forceps to orient the tissue so that it is suspended halfway between the top and bottom of the ring. Mold incubate at four degrees Celsius until the arose has solidified.
Prior to slicing the tissue, prepare a customized wilkinson blade by rinsing with solvents, followed by water. Separate the double blade lengthwise into two single blades. Next, support the Wilkinson blade with a microtone blade.
To do this first, cut the microtone blade to the length of the Wilkinson blade. Then using a Lock Tite adhesive glue the microtone blade to the Wilkinson blade offset from the cutting edge by about three to four millimeters. Now mount the reinforced blade in the vibrating microtome and set the blade angle to 35 degrees.
Prepare the deluxe specimen bath by filling the bath with PBS and packing wet ice around it. Before mounting the tissue embedded in the plug, remove it from the ring mold and block dry the bottom surface. Then glue the dried surface to the mounting disc using Loctite adhesive.
Insert the disc into the vibrator and adjust the section thickness to 50 microns, the speed to two and the blade amplitude to four. While cutting tissue sections, use blunt forceps to transfer each section to a 24 well culture plate filled with half a milliliter of ice cold PBS tween. After slicing, remove most of the aros from around each tissue section using spring scissors prior to beginning the INI hybridization, the sections can be stored for up to 48 hours at four degrees Celsius in PBS tween containing two millimolar sodium azide.
First, make custom sample baskets by cutting the bottom off a micro centrifuge tube at the 100 microliter mark. Heat the cutting of the tube in a flame until the plastic is soft. Then firmly press the soft plastic onto a mesh square.
Allow it to harden and trim the excess mesh. Complete the baskets by using a heated 18 gauge needle to pierce two holes into each tube lid. Make a tray to hold the sample baskets by drilling 24 evenly spaced 12 millimeter holes into the lid of a 24 well plate.
Each hole should be positioned directly over a well before beginning start preheating a covered hybridization chamber filled with half an inch of tap water to 60.5 degrees Celsius. Begin the in situ hybridization by adding two milliliters of PBS tween to each well of the 24 well culture plate. Cover this plate with the prepared lid loaded with baskets.
Now transfer tissue sections into the baskets at room temperature with rocking incubate the sections in 6%hydrogen peroxide with PBS tween, followed by a series of washes with PBS tween, an incubation with proteinase K, another fixation, and more washes. To finish preparing the samples, add two milliliters of the pre-war pre hybridization buffer to each well and incubate the plate inside the heated hybridization chamber for at least one hour. Initiate the hybridization by adding 0.5 micrograms of labeled RIBA Probe to each.
Well then continue the incubation overnight inside the heated hybridization chamber. The next day wash tissues with solution one gradually changing to solution two, then treat with RNAs. Then wash in solution three, followed by washes in TBS tween and tissue blocking buffer while the tissues are incubating in tissue blocking buffer.
Begin a minimum two hour incubation of the anti dig antibody in antibody absorption buffer at four degrees Celsius after the incubation period. Spin down the antibody solution at 10, 000 RPM for a minute. To remove in solubles, add the Senna to two milliliters of antibody solution buffer.
Now remove the tissue from the blocking buffer and incubate them in the prepared antibody solution. Keep them in a hybridization chamber overnight of four degrees Celsius on the third day of the procedure. Wash the tissues several times in TBST with the ole.
Then using a glass pasta pipette. Carefully transfer the tissues into clean micro centrifuge tubes. Wash the tissues for 10 minutes with one milliliter of NTM empty.
Then replace the solution with TMT containing lava sole MBM purple. Place the tubes in the dark and over the next several days, monitor color development and change the solution as needed. After full color development, briefly wash the tissues with TMT containing lava so and then incubate them overnight at four degrees Celsius in PBS containing para formaldehyde.
The next day bleach and wash the tissues. The sections are now ready for imaging. The lower urogenital tract tissue was embedded in agar such that the urethral midline was parallel to the flat surface of the aros plug.
This orientation yielded sagittal sections. Custom made sample baskets protected the delicate tissue sections from dust and particulate matter. During the multi-day in C two hybridization procedure.
They also prevented loss of any sections. It is challenging to limit background staining during the long incubations required to detect low abundance mRNAs. However, the addition of sodium azide and subsequent filtration appears to have limited background staining.
Furthermore, there were no visible differences in background staining when samples were incubated in color development solution for prolonged time periods. Once mastered, this technique can be completed in six days if it is performed properly. Following this procedure, other methods like immunohistochemistry can be performed to determine if the mRNAs expression pattern correlates to the proteins expression pattern.
After watching this video, you should have a good understanding of how to visualize mRNA expression patterns and the developing mouse lower your genital tract.
