The overall goal of the following experiment is to detect accidentally localized mRNAs in adult brain samples. This is achieved by pretreating. The samples by boiling and protease digesting to unmask the mRNA molecules as a second step Hybridization is performed, followed by amplification and detection steps, which enable the visualization of the mRNA of interest.
Next axons are stained with antibodies or dyes in order to verify that the mRNA of interest is localized to axons. The results show the presence of a TF four mRNA in brain samples using different unmasking procedures and counters, staining methods based on microscopy analyses. This method can help answer questions in neuroscience such as which mRNAs are localized and translated in without exons.
Although this method can provide insight into intracon protein synthesis, it can also be applied to other systems such as dendrites and non neuronal cells and tissues in which mRNA localization occurs. After fixing brain slices, according to the instructions in the written protocol, prepare a copin jar containing 60 milliliters of antigen retrieval solution and immerse the slides in the solution. Next, fill a one liter beaker with distilled water and place it in the microwave to buffer the heat.
When performing unmasking, place the coplan jar containing the slides and retrieval solution into the microwave. Boil the slides at high power for five minutes. Immediately after the solution has stopped boiling heat the slides again at high power for an extra five minutes.
Allow the slides to cool at room temperature After cooling, immerse the slides in a dish containing PBS to wash them and then repeat the wash two more times After washing, place the slides on a flat surface and add two to three drops or 100 microliters of DAP B.Probe to two or three brain sections as a control. To assess background staining, add two to three drops of the targeting probe to the experimental sections To detect the RNA of interest, A probe targeting residues 20 to 1381 of mouse a TF four RNA is used here. Use forceps to place param over the slides to avoid probe evaporation.
Then place the slides in the humidified slide box inside the hybridization oven and incubate them at 40 degrees Celsius for two hours. After the incubation time has elapsed, wash the slides in a dish with one x wash buffer for two minutes at room temperature. Next, add two to three drops of amplification reagent a MP one fl to each brain slice.
Then cover with fresh para film place in the slide box and incubate at 40 degrees Celsius for 15 minutes in the hybridization oven. After washing the slides as before, add two to three drops of amplification reagent A MP four fl to each brain slice and cover with perfil. Incubate the slides in the slide box at 40 degrees Celsius for 15 minutes in the hybridization oven.
Wash the slides twice with one x wash buffer for two minutes each at room temperature. As before following the last wash cover each slice with 100 to 200 microliters of blocking solution containing three milligrams per milliliter BSA 100 millimolar glycine and 0.25%Triton X 100 in PBS cover each slide with paraform and incubate for 30 minutes At room temperature following the incubation, add 100 to 200 microliters of antibody diluted in blocking solution to the slides. An anti choline acetyl transferase antibody is used here after covering the slides with perfil incubate for two days at four degrees Celsius.
Make sure that brain slices don't dry out if needed, reapply anti CHATT antibody solution to brain slices 24 hours after incubation. After washing the slides three times in one XPBS for five minutes. At room temperature, add 100 to 200 microliters of the appropriate Alexa conjugated secondary antibody to the slides.
Cover with param and incubate for one hour at room temperature while protected from light after the hour has elapsed. Wash the slides three times in one XPBS for five minutes each as before, and then wash once with distilled water. Mount the slides with mounting medium containing DPI and then visualize the brain slices under the fluorescence microscope.
After preparing formal and fixed paraffin embedded human brain samples, according to the instructions in the written portion of the protocol, perform heat induced antigen unmasking by immersing the slides in hot pre-treat two solution and boiling for 15 minutes. After washing three to five times in distilled water and three to five times in fresh 100%ethanol, allow the slides to dry for five minutes at room temperature to perform protease induced antigen unmasking. Add four drops of pretreat three cover with perfil and incubate for 30 minutes at 40 degrees Celsius in the hybridization oven After washing three to five times in distilled water.
As before, add four drops of the DAP B probe set to control brain slices to assess background staining and four drops of the targeting probe set to the experimental sections. Place the pair of film covered slides in the slide box and incubate for two hours at 40 degrees Celsius in the hybridization oven. After following the steps in the written protocol to develop the DAB signal for the mRNA of interest, check for the presence of a signal under a brightfield microscope.
Define reference areas in the brain samples in which to monitor the presence of puncta throughout the counter stain procedure. To counter stain. First place the slides in luxal fast blue preheated to 60 degrees Celsius and incubate for 30 minutes at 60 degrees Celsius.
After the incubation, rinse the slides several times in distilled water and then dip the slides in 0.05%lithium carbonate solution several times to start differentiation. Next, dip the slides twice in fresh, 75%ethanol and rinse in distilled water. Check the brain slices under a bright field microscope.
Note that gray and white matter should start to be distinguishable and RNA granules still visible as dark blue black puncta. Verify that axons start appearing as light blue fibers repeat the luxal blue staining procedure incubating with luxal blue in 10 to 20 minute steps and carefully monitoring the counter stain and the presence of RNA granules when optimal staining has been achieved. Place the rinse slides in crestal violet solution and incubate for 10 minutes following the incubation.
Rinse the slides in distilled water. Dip the slides in 70%ethanol five to 10 times and then dehydrate through three quick changes of 100%ethanol in a fume hood. Clear brain slices by incubating twice in xylene.
Alternative clearing agent for two minutes and a third time for five minutes. At room temperature mount the brain slices in xylene based permanent mounting medium and analyze under a bright field microscope. Fish was performed using protease induced unmasking followed by antibody detection of choline acetyl transferase shown in red.
The antibody failed to recognize chat when protease treatment was performed. Examples of results obtained with a non-target probe and the a TF four targeting probe using the same microscope settings and image adjustments are shown. Green ATF four positive granules with unclear axonal localization are indicated with question marks.
Blue areas are dappy stained nuclei when fish was performed using heat induced antigen. Unmasking chat immunofluorescent staining again shown in red was successful. A TF four positive granules localized to axons appear orange and are marked as okay following ish axons were counterstain with luxal.
Fast blue and neuronal SOTA were counter stained with crestal violet suboptimal luxal. Fast blue staining can result if the temperature is decreased. Here the samples were stained with luxal fast blue at 40 degrees Celsius for four hours.
A TF four positive granules with unclear accidental localization are indicated with question marks. Suboptimal staining also occurs when the intensity of the counter stain is not checked after short incubation periods as seen here. Examples of optimal LFB staining combined with ish using a non-target probe or the a TF four targeting probe are shown here.
Image acquisition was automatically adjusted for the best signal to noise ratio. Axon localized a TF four granules are marked as okay. Once mastered.
This technique can be done in one to three days depending on the counterstain method chosen After it's development. This technique paves away for neuroscientists to explore mRNA translation and localization in nuance.
