Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This procedure is highly sensitive and allows a visualization of several transcripts with minimal background. This method can generate accurate maps of the transcriptional profile of highly heterogeneous cell types. Demonstrating the procedure will be me and Johnson Bob-manuel, a research technician in the laboratory.

Begin by collecting the entire vagal ganglionic mass from the eight weeks old mouse from each side, with the help of a dissecting scope and surgical instruments. After decapitation of the mouse, use small forceps to remove the sternohyoid and the omohyoid muscles lateral to the trachea to expose the occipital bone. Clear the whole region of muscle and tissues until the carotid artery, the vagus and hypoglossal nerve and foramen magnum are visible.

Then cut the entire hypoglossal nerve with small spring scissors. Locate for the nodose ganglion as a translucent mass close to the foramen magnum. Using small scissors, carefully break the occipital bone.

To expose the jugular ganglion further, locate black melanocytes at the surface of the jugular as a visual landmark. When the ganglions are found, cut the nerve attachments between the vagal ganglionic mass and gently pull on the peripheral end of the vagus nerve while simultaneously cutting the jugular ganglion towards the brainstem with small spring scissors for the extraction of the whole ganglionic mass. Remove the conjunctive tissue, ganglionic mass and fat sticking to the vagus nerve.

Keeping approximately a half centimeter of the vagus nerve to facilitate handling and localization of samples. Place the remove to ganglia with the help of fine forceps in 1.5 millimeters micro centrifuge tubes. And incubate at four degrees celsius in formalin for 24 hours.

And then with 30%sucrose for 24 hours. After incubation, position the ganglia inside a drop of a four millimeter diameter of optimum cutting temperature medium on a piece of aluminum foil. And phrase the sample on a bed of dry ice.

Cut the frozen samples with a cryostat into sections of 14 micrometer thickness at minus 20 degrees Celsius and collect the cut sections on the glass microscope slides as three series 42 micrometers apart. Rinse the slides with tissue sections in PBS. And bake for 30 minutes at 60 degrees Celsius in a baking oven.

Then, postfix the slides in 4%formalin for 15 minutes at four degrees Celsius. Serially dehydrate the slides in 50%70%and 100%ethanol for five minutes each. And apply hydrogen peroxide solution to the samples for 10 minutes before rinsing in double distilled water.

For In situ hybridization, or ISH, treat the samples with the target retrieval reagent for five minutes. Then sequentially rinse the samples in double distilled water and 100%ethanol followed by air drying. Using a hydrophobic pen, create a barrier around the samples.

While the samples are being treated with protease for 30 minutes at 40 degrees Celsius and a hybridization oven, prewarm the probes at 40 degrees Celsius and cool them at room temperature before use. After moving from the oven, incubate the slides with the desired combination of target probes for two hours at 40 degrees Celsius in a hybridization oven. Incubate negative control tissue with the dihydrodipicolinate reductase or DapB, target C1 probe for two hours at 40 degrees Celsius.

Rinse the slides in wash buffer and store in five times saline sodium citrate at room temperature overnight. The next day, rinse the slides with wash buffer before incubation with amplification reagents. And treat with channel specific reagents as described in the manuscript.

Wash the slides followed by treatment with DapB for 30 seconds. Then immediately apply the mounting medium on each slide and place a cover slip over the tissue section. Keep the tissues horizontal in a slide holder at four degrees Celsius until imaging.

Prepare a confocal microscope for imaging by setting the required parameters. Acquire the images with a line averaging of four and a pixel size of at least 1024 by 1024 to improve the quality of the images. Once satisfied with the acquisition parameters, start acquiring the images using the same settings.

Attribute false colors to each channel to facilitate visualization. Using the digital images, perform cell counting by identifying the outline of RNA scope positive profiles with one nucleus lightly stained with DapB. Calculate the percentage of RNA scope positive profiles expressing each transcript.

The optimal acquisition parameters were chosen for each laser based on the unspecific fluorescence obtained in the negative control tissue. The endogenous fluorescence in the nodose ganglion of a young adult mouse is minimal. And incubation with a DapB probe, it did not result in signals.

At increased power laser and gain with the 488 nanometer laser, unwanted background and random fluorescent dots appear. The RNA scope technique was applied to detect three genes in tissue sections of the vagal ganglionic mass. The tissue profile was positive for GHSR and CCK1R.

The profile included both Phox2b and CCK1R transcripts in the rostral part of the nodose ganglion. As a result of the multiplex ISH in nodose afferent neurons, Phox2b signals specifically accumulated in afferent neurons, located in the nodose ganglion. CCK1R signals intensely labeled many neurons in the nodose ganglion.

At low magnification, cells expressing low CCK1R or GSHR signals could not be observed easily. DapB helped to visualize the tissue and identify vagal afferent neurons with a large and lightly stained nucleus. At high magnification, Phox2b positive profiles are evident.

Profiles two and three are Phox2b cells with high and moderate CCK1R signals. Profile four expressed both GHSR and CCK1R. As a result of the multiplex ISH of the jugular afferent neurons, the PRDM12 transcript could be seen specifically accumulated in afferent neurons located in the jugular ganglion.

At high magnification, moderate signals for CCK1R were detected in profiles one and two. Profile three is a representative of PRDM12 cells negative for CCK1R or GHSR. Consistency of dissection is an important factor.

It is equally important to verify that the imaging acquisition parameters do not generate unwanted background signals. Multiplex In situ hybridization has become the gold standard in molecular established, especially in the field of neuroanatomy. This method can be easily combined with immunohistochemistry.