Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

The overall goal of this experiment is to simultaneously detect the expression of three different genes in brain sections, using double fluorescence in situ hybridization followed by immunostaining. This method can help answer key questions in the neuroscience field, such as which cell type expresses my gene of interest? The main advantage of this technique is that you don't need a good antibody for your gene of interest to be able to localize its expression to a specific cell type.

To summarize this procedure, on the first day, fixed tissue sections are cut and hybridized with two differently-labeled RNA probes overnight. On the second day, the sections are incubated overnight with an antibody targeting the FITC labeled probe. This antibody is conjugated with horseradish peroxidase, which catalyzes the addition of a fluorescent Tyramide on the third day.

Then, an alkaline phosphatase conjugated antibody is used to target the DIG labeled probe. On day four, this enzyme catalyzes the fluorescent precipitate formation when incubated with Fast Red solution. Then the sections are incubated overnight with a primary antibody targeting a protein of interest.

Finally, on day five, this protein is visualized with a fluorophore conjugated secondary antibody. To begin this procedure, choose a DNA clone that contains the cDNA sequence of the gene of interest in a plasmid with RNA polymerase promoters. Next, grow the DNA clone, extract the plasmid with a small scale plasmid purification kit, and sequence it with a T7 and SP6 universal primers.

Digest 10 to 20 micrograms of plasmid DNA with a restriction enzyme that cuts at the five prime end of the sense strand of the cDNA. Incubate it for 1.5 hours at 37 degrees Celsius. Then run a small aliquot on an agarose gel to check that the plasmid is fully linearized.

To purify the linearized plasmid, add 1/10 of the volume of three molar sodium acetate, one volume of 10 millimolar Tris-HCl equilabrated phenol, and one volume of chloroform isoamyl alcohol. Centrifuge at 16, 000 Gs for one minute at room temperature, and extract the upper aqueous phase. Then add one volume of chloroform IAA.

Centrifuge it at 16, 000 Gs for one minute, and extract the upper aqueous phase. Repeat this step once again. Then, add two volumes of ethanol, and keep it at negative 20 degrees Celsius for one hour, or overnight.

After that, centrifuge at 16, 000 Gs at four degrees Celsius for 10 minutes. Discard the supernatant, and wash the pellet with 70%cold ethanol. Then re-suspend it in TE at a concentration of one microgram of cDNA per 2.5 microliters.

To synthesize the two probes, first select the appropriate RNA polymerase. Then, prepare the in vitro transcription reaction, and bring the final volume up to 20 microliters with DEPC-treated water. Incubate at 37 degrees Celsius for 1.5 hours.

Subsequently, run one microliter of the transcription reaction on a 1%agarose gel to check if the reaction has worked. The gel should show the linear template and a bright band of the probe. In this procedure, cut 15 micrometer thick sections of frozen tissue in a cryostat.

Collect the sections onto the microscope slides, and leave the slides to dry for one hour to ensure the tissues adhere to the slides. The most important factor for this procedure to work well is the quality of the sections. This depends partly on having well-perfused and fixed tissue, and partly on sectioning technique to obtain flat sections that are free of RNase contamination.

Then, 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 cover slip. 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 Celcius with a horseradish peroxidized 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 tiromide to the slides and leave them for 10 minutes at room temperate. 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 four degrees Celcius 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 Celcius 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. To perform immunohistochemistry, incubate the slides with IHC blocking buffer for one hour at room temperature in a humidified chamber. Next, incubate the slides in the primary antibody solution at four degrees Celsius overnight.

The next day, wash the slides with PBST for 10 minutes three times. Incubate them in the secondary antibody solution at room temperature for one hour. Then, wash them with PBST three times for 10 minutes each time.

Subsequently, partially dry the slides at room temperature and mount with a fluorescence mounting medium. Leave the slides to dry before imaging them in a confocal microscope. Shown here are the representative images from ISH for Aspa and Mbp, followed by immuno-staining for OLIG2 combined with hook-staining.

Aspa was expressed in some MBP-positive OLIG2-positive cells in the cortex and in the corpus callosum. Some Mbp-positive OLIG2-positive cells do not express Aspa. While performing this procedure, it is important to remember to keep the slices and all the solutions free of RNase contamination.

After watching this video, you should have a good understanding of how to study gene expression patterns by N-citrine inhibization and immunohistochemistry. This technique can be adapted for a variety of tissue of different origins and developmental stages.