This article details a method for detecting mRNA expression in oligodendrocytes using fluorescence microscopy. The technique involves hybridization of RNA probes to specific mRNA targets, followed by signal amplification and visualization.
Take a chemically-fixed mouse brain section.
Add a hybridization mixture containing a permeabilization agent and RNA probes labeled with FITC or DIG.
Incubate to allow cell permeabilization and probe entry.
The probes bind to complementary sequences on MBP mRNA, a reference marker ubiquitously expressed in oligodendrocytes, or ASPA mRNA, the target.
Wash to remove unbound probes.
Add a blocking buffer to prevent non-specific antibody binding.
Introduce HRP-conjugated anti-FITC antibodies targeting the FITC-labeled probe.
Wash, then add FITC-tyramide.
HRP oxidizes FITC-tyramide into a highly reactive form that binds to nearby proteins, amplifying the fluorescence signal.
Wash and reintroduce the blocking buffer.
Add alkaline phosphatase-conjugated anti-DIG antibodies targeting the DIG-labeled probe.
Wash and apply the Fast Red solution.
Alkaline phosphatase dephosphorylates Fast Red, producing a fluorescent precipitate.
Visualize under a fluorescence microscope.
Co-localization of Mbp and ASPA signals indicates ASPA expression in oligodendrocytes.
Dilute the two probes in hybridization buffer, pre-warmed to 65 degrees Celsius, and mix well. Apply 300 microliters of hybridization mixture onto each slide, and cover with an oven baked coverslip. Subsequently, incubate the slides overnight at 65 degrees Celsius in a humidified chamber.
The next day, transfer the slides into a Coplin jar containing pre-warmed wash buffer, and wash the slides for 30 minutes, twice at 65 degrees Celsius. After that, wash the slides for 10 minutes three times with MABT wash solution at room temperature.
In this step, use a hydrophobic pen to draw circles around the tissue sections. Then, incubate the slides for one hour at room temperature with ISH blocking buffer in a humidified chamber. Next, incubate the slides overnight at 4 degrees Celsius with a horseradish peroxidase-conjugated anti-FITC antibody.
Then, place the slides into a Coplin jar containing PBST. Wash them in PBST three times for 10 minutes each time, and replace with fresh PBST each time. Afterward, add fluorescent tyramide to the slides, and leave them for 10 minutes at room temperature. Place the slides into a Coplin jar, and wash in PBST for 10 minutes three times.
Subsequently, incubate the slides with ISH blocking buffer for one hour at room temperature. Then, incubate the slides overnight at 4 degrees Celsius with an alkaline phosphatase conjugated anti-DIG antibody. After that, wash them with MABT three times for 10 minutes each time. Next, wash the slides twice with 0.1 molar Tris-HCL at pH 8.2 for five minutes at room temperature.
Filter the fast red solution, and incubate the slides at 37 degrees Celsius with fast red solution in a humidified chamber. As the time for optimal development varies, check the slides regularly with a fluorescent microscope to monitor the formation of precipitate. Then, wash them three times with PBST for 10 minutes each time.
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