Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol combines fluorescence RNAscope in situ hybridization and immunohistochemistry using fresh-frozen or fixed brain preparations. This technique allows flexibility by demonstrating a combination of highly sensitive in situ hybridization and immunohistochemistry methods for spatial visualization of RNA and protein targets in the same tissue specimen. This method is applicable for multiplex labeling in brain tissue section, aiding the investigation of spatial organization and functional significance of mRNA and proteins within heterogeneous neuronal populations in neuroscience.

This protocol mainly focuses on processing steps for fresh-frozen tissue and paraformaldehyde fixed tissue. To begin sectioning the fresh-frozen brain tissue, affix it to the pre-chilled cryostat chuck and cut 14 micrometer thick coronal sections. Mount the section onto a charged glass microscopy slide.

Transfer the mounted slide into a slide box. Then transport the box to the minus 80 degree Celsius freezer on dry ice. For tissue fixation, filter the prepared paraformaldehyde solution.

Cool it to 4 degrees Celsius. Bring the mounted slide from the minus 80 degree Celsius freezer on dry ice and immediately immerse it in the pre-chilled paraformaldehyde solution for 15 minutes, Perform dehydration of fresh-frozen tissue to ensure that the glass slide is completely dry. After removing the brain from a transcardially profusion fixed mount, using a vibrating microtome, cut 30 micrometer thick tissue sections.

Store the cut sections in cryoprotectant solution. On the day of the FISH assay, transfer the free floating section into a 12-well cell culture plate containing 0.1 molar PBS and agitate it at 90 to 100 RPM on a rotating platform shaker to wash off cryoprotectant. Then, using a paintbrush, mount the section flat on a glass microscopy slide to prevent detachment during washes, and air dry for at least two hours.

Using a hydrophobic barrier pen, draw a hydrophobic barrier around the section to contain the FISH reagents. Following pre-processing, the tissue is incubated in protease solution and hybridized with RNAscope probes for two hours as per the manufacturer's guidelines. Then, sequential amplification steps and fluorescence immunohistochemistry are performed.

Start by preparing a humidified light protected chamber for incubating slides. In a water bath, warm 50X wash buffer and probes to 40 degrees Celsius for 10 minutes. Once cooled to room temperature, prepare one liter of 1X wash buffer from the 50X stock concentration.

Prepare the probe mixture as described in the text manuscript. For proteinase treatment, add proteinase 3 on to the tissue section and incubate it at room temperature for 30 minutes. To wash off the proteinase, gently immerse the slide in 0.1 molar PBS at room temperature, avoiding section dislodging.

For hybridization, add the probe mixture on to the tissue section. Place it in the humidified pre-warmed chamber. Then incubate it for two hours at 40 degrees Celsius.

Rinse the tissue section with wash buffer twice for two minutes. For signal amplification, using a dropper bottle, add an amplification solution to cover the tissue section and incubate as described in the text. Add the blocking solution on to the tissue section and incubate for one hour at room temperature to prevent non-specific binding.

Flick the slide to remove the excess blocking buffer after incubation. Now, incubate the section with primary antibodies overnight at 4 degrees Celsius. The next day, wash the slide three times with 1X TBS.

Add the secondary antibody and incubate the sections for two hours at room temperature. Examine the slide under an epifluorescence microscope equipped with a camera. Acquire representative images at 20 times magnification and save them as TIF files.

In the present study, the tissue quality, integrity, and the absence of bacterial contamination were confirmed by the absence of DAPI labeling. Labeling from positive control probes targeting ubiquitin C, peptidylprolyl isomerase B, and RNA polymerase IIA mRNA confirmed RNA integrity. In fresh-frozen preparations to distinguish GalR1 mRNA positive neurons within the NTS, additional neurochemical markers were used.

The membrane-bound vesicular transporter was clearly labeled. In contrast, cytoplasmic proteins such as tyrosine hydroxylase and GFP expressed under the control of PHOX2B promoter were faintly observed and described as flocculent as they lack a clear outline. GalR1 FISH probe labeling of cytoplasmic GalR1 mRNA was punctate and clearly observed.

To confirm that immunohistochemistry quality depends on protein subcellular localization, different nuclear and cytoplasmic proteins were labeled in parallel with FISH in the same tissue sections. The cytoplasmic PHOX2B GFP had a flocculent appearance. Whereas the nuclear PHOX2B protein signal was clear, which confirmed higher quality immunolabeling when combined with FISH.

When performed on fixed frozen sections in combination with FISH, immunohistochemistry was reliable irrespective of subcellular localization. GlyT2 mRNA positive neurons were located ventral to the NTS and not within the NTS. GlyT2 mRNA positive and PHOX2B mRNA positive neurons did not colocalize.

A subpopulation of PHOX2B mRNA positive NTS neurons was tyrosine hydroxylase immunoreactive, and none contained GlyT2 mRNA. RNAscope enables visualization and analysis of RNA distribution at single molecule resolution in cells and tissues. It empowers research in novel RNA species, disease mechanisms, gene expression patterns, neuron diversity, and cellular interactions.