This method can help answer key questions in the field of alternative pre-mRNA splicing. Specifically it provides a robustly, sensitive assay system to examine alternative splicing patterns in situ in tissue slices. With this method, alternative splicing can be analyzed simultaneously in many subtypes that are contained in complex biological structures such as a mouse brain.
The main advantage of this technique is that it uses shock anti-static exon junction probes in mRNA in situ hybridization to produce isoform specific hybridization signals. Building signal amplification technology increases the sensitivity of the detection to provide robust hybridization signals. The signals detected was exon inclusion escaping specific probes that are used to calculate the percentage splice in vitals at different anatomical areas of a mouse brain.
In this study NF1 exon 23a is used as an example to illustrate the use of the assay system. However, the system can be adapted to analysis expression patterns of many alternative exons. To start mouse brain section D paraffinization, fill two washing dishes with 250 milliliters of xylene.
And another two with 250 milliliters of 100%ethanol inside a fume hood. Insert the tissue slides into a washing rack. Next, place the washing rack in a xylene containing dish and incubate for five minutes while moving the rack up and down at least three times and repeat with the second xylene dish.
Then place the rack in an ethanol containing dish and incubate for two minutes with the same up and down movements and then repeat with the second ethanol dish. To dry the slides, move the rack from the second ethanol dish and place it inside an incubator at 60 degrees Celsius for five minutes. Use a hydrophobic barrier pen to draw a 0.75 inch by 0.75 inch barrier around the tissue section and air dry for three minutes at room temperature.
Turn on the hybridization oven and set the temperature at 40 degrees Celsius. After completely wetting humidifying paper with distilled water, place it in a humidity control tray. Then insert the humidity control tray into the hybridization oven.
After placing the dried slides on a bench, completely cover the tissue sections on each slide with five to eight drops of hydrogen peroxide solution. Incubate for 10 minutes at room temperature. Tap the slides on an absorbent paper, one at a time to remove the hydrogen peroxide solution and insert the slides into a washing rack.
Immerse the rack in a washing dish containing water while moving it up and down three to five times to wash the slides. Fill a one liter glass beaker placed on a hot plate with 700 milliliters of 1X target removal treatment and cover it with foil. Insert a thermometer through the foil cover and turn on the hot plate, set on high temperature for 10 to 15 minutes.
Once the temperature of the retrieval reagent reaches 98 degrees Celsius, set the hot plate at low temperature to maintain a mild boiling condition. Next, carefully remove the foil and place the rack inside the beaker. Cover the beaker with foil and incubate for 15 minutes.
Use forceps to transfer the rack from the beaker to a washing dish containing 200 milliliters of distilled water and incubate for 15 seconds. After 15 seconds place the rack in a washing dish containing 100%ethanol and incubate for three minutes. Remove the rack from the dish and dry at 60 degrees Celsius for 10 minutes.
After placing the slides on a stain rack, cover the tissue sections with five drops of protease 3 per slide. Carefully place the rack in the humidity control tray and insert the tray into the hybridization oven and incubate at 40 degrees Celsius for 30 minutes. After removing the rack from the tray, tap the slides to remove excess liquid and insert them into a washing rack.
Place the rack into a washing dish containing distilled water and move the rack up and down three to five times to wash the slides. To prepare for in situ hybridization, incubate the probes and AMP reagents at room temperature for 30 minutes. Insert the humidity control tray into the hybridization oven and set at 40 degrees Celsius.
For probe hybridization, remove the slides from the washing rack and tap them to remove excess liquid. Place the slides on a stain rack. Use a pencil to label the slides with the probe name.
Place three adjacently cut brain tissue slides to be hybridized with three different neural fiber mitosis type one probes together. Because there are very different isoform specific probes, I use adjacently cut tissue sections. It is critical that these sections are treated exactly the same in hybridization, amplification, and signal detection steps to ensure the accuracy of the percentage splice in calculation.
Cover the tissue sections with four drops of probe per section, one slide at a time to prevent drying out of the sections. Then place the stain rack in the humidity control tray and insert it into the 40 degrees Celsius oven for two hours. Next, place a washing rack in a washing dish filled with 200 milliliters of 1X wash buffer.
Then tap each slide, one at a time on a paper towel to remove excess liquid. And immediately insert each slide into the washing rack. Incubate at room temperature for two minutes in a dish filled with fresh wash buffer while moving the rack up and down three to five times and then repeat the wash in a new dish.
For signal amplification, tap each slide to remove excess wash buffer and place them on a stain rack. Cover the tissue section on each slide with four drops of AMP zero reagent. Next, place the rack in the humidity control tray, insert the tray into the oven and incubate at 40 degrees Celsius for 30 minutes.
Wash the slide twice with wash buffer as previously done in this experiment. Repeat the signal amplification step with AMP reagents one through six as described in the text protocol and wash the slides after each step. For signal detection, remove excess wash buffer from the slides by tapping and place them on a stain rack.
Next, mix two microliters of fast red B with 120 microliters of fast red A in a micro centrifuge tube. Add the solution to the tissue sections on each slide to fully cover them. Place the stain rack in the humidity control tray, cover the tray and incubate for 10 minutes at room temperature.
Then remove the solution and insert the slides into a washing rack. Place the rack in a washing dish containing tap water to wash the slides and repeat the wash. After the second wash, tap on an absorbent paper to remove excess water.
Next, place the rack in a dish containing 50%hematoxylin staining solution. Move the rack up and down several times. And incubate for two minutes at room temperature.
Transfer the rack to a washing dish containing tap water. Move the rack up and down five times and repeat the wash until the slides become clear, while tissue sections remain purple. Then transfer the rack to a dish containing 0.02%ammonia water.
Move the rack up and down two to four times until the sections turn blue. Wash the slides with tap water three to five times in the staining dish. Next, place the rack inside an incubator and incubate for 15 minutes at 60 degrees Celsius to dry the slides.
When completely dry, add one drop of mounting medium on each slide to cover the slice. Then, carefully place a 24 by 50 millimeter coverslip over the tissue sections on each slide. After air drying the slides for 30 minutes at room temperature, proceed with data collection.
To calculate the percent spliced in or PSI value for exon 23a, the number of cells and dots in three sub-regions of cortex, and hippocampus CA3 are counted. RNA in situ hybridization assay is performed in brain sections of CD1 wild type mice using three different NF1 specific probes. Red punctate dots observed in the cortex and hippocampus sections, indicate positive signals by NF1 total, inclusion specific, and skipping specific probes.
The ISH signals from the three probes are then counted and used to calculate PSI. RNA in situ hybridization assay is performed in the cortex of C57 black 6J wild type mice and C57 black 6J mutant mice in which exon 23a is included in all cell types. Red punctate dots observed in the cortex of C57 black 6J wild type mice, indicate positive signals by NF1 total, inclusion specific, and skipping specific probes.
In C57 black 6J mutant mice, similar signals are obtained by NF1 total and inclusion specific probes. However, no signal is detected when skipping specific probe is used, indicating that two probes targeting exon inclusion or skipping are highly specific. Because multi-color labeling on the same tissue slides is not yet available for this procedure, different probes are used on the different tissue slides.
To obtain the accurate percent splicing values, it's important to use adjacently cut slides and treating slides in the exact same way throughout this procedure. Complementing this procedure are the measures like immunohistochemistry, can be performed to answer additional questions regarding the localization and function of differentially expressed splicing isoforms.
