The overall goal of the following experiment is to locate and quantify micro RNA expression within kidney tissues. This is achieved by first hybridizing, a diggen and tagged probe to a given micro RNA within the kidney tissue. As a second step, an antibody to digo genin, which has a conjugated alkaline phosphatase, is incubated with the tissue.
Next, an N-B-T-B-C-I-P substrate is added to the tissue in order to visualize the location of the micro RNA through development of a blue colored product at sites of the hybridized micro RNA probes results are obtained that show where microRNAs are expressed in kidney regions and nephron segments based on hybridization of micro RNA complimentary probes. This procedure uses formal and fixed paraffin embedded kidney sections that are caught onto microscope slides at a thickness of five microns. To prepare the kidney sections for analysis, first remove the paraffin from the tissue by three washes in fresh xylene for five minutes.
Each rehydrate the tissue sections by five minute incubations in decreasing concentrations of ethanol in doubly distilled water. Next, treat the slides with enough dathyl, percarbonate, or desy to completely cover the tissue sections for one minute. Make sure the tissues do not dry out.
Remove the desy and rinse the slides in. Dessy treated PBST twice for five minutes. Make sure the dessy containing waste is collected for proper disposal.
From this point on, all reagents should be dsy treated To eliminate RNAs, add proteinase K to prewarm proteinase K buffer to a final concentration of 10 micrograms per milliliter. Cover the tissue sections with the protein A K solution and incubate at 37 degrees Celsius for five minutes. Quickly remove the proteinase K solution and add 0.2%glycine in PBST for 30 seconds.
Rinse the slides in. Desy treated PBST twice for 30 seconds each. Lastly, fix the tissue sections in freshly made 4%para formaldehyde for 10 minutes.
To begin this procedure, add 50 microliters of hybridization buffer per tissue section and cover with RNAs free hybrid slips that are caught just larger than the tissue sections. Place the slides inside a chamber humidified by lab wipes soaked in 50%form amide 50%five XSSC pre hybridized the tissue sections by placing the humidified chamber for two hours in an oven at the hybridization temperature determined for the three prime dig oxygen and labeled probe. The hybridization temperature for the probe is usually the DNA melting temperature or TM of the probe minus 21 degrees Celsius.
For example, the hybridization temperature for the mere 3 82 probe is 54 degrees Celsius because its TM is 75 degrees Celsius. Prepare the micro RNA probe and the positive and negative controls by diluting each to 40 nano molar in hybridization buffer. The positive control is the U six small nuclear RNA and the negative control is a scrambled sequence.
Heat the probe and the controls at 65 degrees Celsius for five minutes. Remove the slides from the hybridization oven. Remove the hybrid slips and excess hybridization buffer from the pre hybridization setup.
Add 75 microliters of probe containing buffer directly onto each tissue section. Cover with hybrid slips that are, again, just large enough to cover the tissue sections. Keeping the slide holder level.
Return the slides to the hybridization oven at the appropriate temperature. Incubate overnight for approximately 16 hours. Once hybridization is complete, remove the slides from the hybridization oven.
Add 200 microliters of two XSSC heated to the hybridization temperature just under one edge of the cover slip to help release the hyper slip from the tissue. Remove the hyper slip by tilting the slide. After the cover slips have been removed, wash the slides in 200 microliters of 50 percentfor amide 50%two XSSC at the determined hybridization Temperature three times for 30 minutes each.
Rinse the slides five times in PBST at room temperature for five minutes each on an orbital shaker at low speed. Begin this procedure by incubating the slides in blocking buffer for one hour at room temperature on an orbital shaker at low speed. After one hour, add 100 microliters of anti dig AP FAB fragments to each tissue section cover with hybrid slips.
Cut just large enough to cover the section. Incubate the slides in a humidified chamber at four degrees Celsius overnight or for approximately 16 hours the following day. Wash the slides seven times in PBST at room temperature for five minutes each time on an orbital shaker set at low speed.
Next, wash the slides in AP buffer three times for five minutes each. Prepare the N-B-T-B-C-I-P developing solution by adding 200 microliters of N-B-T-B-C-I-P solution to 10 milliliters of AP buffer. Add 400 to 500 microliters of the diluted N-B-T-B-C-I-P solution to each slide to completely cover all tissue sections.
Develop in a dark level humidified chamber for four to five hours at room temperature when the chole metric detection is complete. Dehydrate the sections in increasing concentrations of ethanol for five minutes each clear the sections through five incubations in xylene for five minutes each time. Lastly, mount the slides in per mount mounting medium and dry them overnight.
Ensuring that the tissue section does not dry out is critical to the success of this insi hybridization protocol. The left part of this figure shows a portion of a kidney section in which the hybrid slip slipped off of the edge of the tissue, allowing it to become partially dehydrated. Despite rehydration and coverage in the remaining steps, the signal in the dehydrated portion is artificially high compared to the portion that remained covered by the hyper slip.
The calibration bar represents 100 microns in this image. The importance of optimizing the duration of N-B-T-B-C-I-P development is demonstrated in these next figures. Panel A shows that development periods of longer than four to five hours results in pronounced nonspecific color development and scrambled micro RNA control probed tissues throughout the kidney.
Panel B shows that incubations longer than four to five hours do not result in further improvement in signal when kidneys are probed for high abundance targets such as the U six small nuclear RNA control. These figures adapted from a previous study confirm the applicability of this in situ hybridization method to micro RNA detection in kidney tissue panel A shows representative images of mere 3 82 expression obtained by insi hybridization in kidney sections from mice with or without unilateral ureteral obstruction or UUO and treated with either anti-ME 3 82 or antis. Scrambled UUO induced an increase in mere 3 82 expression as shown by the darker staining in the UUO plus antis scrambled tissue compared to the sham plus antis scrambled tissue.
This increase was blocked by treatment with anti-ME 3 82. The graphs in panel B show mere 3 82 expression of sham plus antis scrambled treated mice as percent average integrated optical density. A measure of pixel intensity as observed, UUO induces a significant increase in mere 3 82 expression in the outer and inner medulla and anti-ME 3 82 blocks.
This increase, all of which can be visualized and measured by micro RNA in situ hybridization. These results are also consistent with results from Q PCR R that are not shown here, indicating that the use of INI two hybridization to detect mere 3 82 expression in formal and fixed kidney is sensitive enough to provide quantifiable results that mirrored qPCR R based expression analysis. Additionally, in C two hybridization allows the measurement of distinct regional differences in micro RNA expression within the kidney.
