Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Transcript

This method can help to answer key questions about tick entomology, such as assessing the presence of viable bacteria in the tick spatially and temporally. The main advantages of this technique are that it does not require sectioning of the ticks or expensive equipments, and that it allows spacial and temporal visualization of gene transcripts. Generally, individuals new to this technique will struggle because it involves multiple steps and reagents.

However, this technique is not laborious. Although it can take about three days to complete, hands on time is only a few hours each day. In preparation, feed the collected ticks for 48 to 72 hours before the gut collection.

To dissect the gut from the tick add 20 to 30 microliters of MEMFA solution to a clean glass slide. Then place the tick into the MEMFA, and adjust focus to the tick's head. Now, using a pair of sterile high carbon steel razor blades slice off the head region at the scutum leaving the body of the tick in tact.

Next, gently press the body to push the gut diverticula and salivary glands out of the body cuticle. Then without damaging the guts, carefully separate them from the salivary glands and carefully scoop the cuts onto a razor blade. Collect all of the harvested guts in a 1.5 milliliter tube in 500 micrometers of MEMFA.

Then incubate the tube at room temperature with gentle rocking for one hour. The next day dehydrate the tissue as indicated in the text protocol and store it at minus 20 degrees Celsius. To prepare a mesh basket, first, use a heated single edge blade on a scalpel to cut the bottoms off of a two milliliter or five milliliter snap top centrifuge tube.

The blade may need to be reheated several times before the cut is completed. Next, cut a square of a 110 micron nylon mesh that is slightly larger than the new openings. Then, bond the mesh to the opening by lightly pressing the tube into the mesh on a hotplate set to medium/low heat until a good seel is made.

After it cools, trim off the excess mesh, and make sure there are not gaps. Make 24 of these baskets to match a 24 well plate. Next, cut holes into the cover of a 24 well plate, such that the lip of the sample basket will grip the hole and not fall through.

When completed, the assembled sample baskets should sit all the way into the wells. Now, use this fitted basket holder to transfer baskets from one plate to another with relative ease all through the in situ hybridization. Use two such plates so that while the sample is incubating in one, the other can get prepared for the next wash.

This section covers certain steps of the in situ hybridization. See the text protocol for a complete description. To begin, transfer each fixed tissue sample to a labeled sample basket.

Organize the plate by experimental conditions, and stain at least five guts per condition. First, gently rehydrate the samples. Avoid introducing air bubbles by gradually increasing the ratio of PBS-T to ethanol in each wash.

During the first day, after the two hour pre-hybridization step, instead of discarding the hybridization buffer, store it for future use during the assay. Later, when treating the samples with probe hybridization solution, cover the plate snugly with aluminum foil to prevent evaporation of the solution. On the second day, load the reserved hybridization buffer into the 24 well plate, and warm the plate up to 60 degrees Celsius.

Later that day, to remove all traces of probe and hybridization buffer, wash the samples twice with 2x SSC for three minutes per wash. Then wash the samples three times with 2x SSC for 20 minutes per wash at 60 degrees Celsius. Then prepare the primary antibody in blocking buffer.

Load 500 microliters of this mixture into each well. After the blocking step, incubate at room temperature for about four hours or at four degrees Celsius overnight. On the third day, run the color reaction in chromogenic substrate.

Cover the sample plate with aluminum foil. And during the reaction, periodically check for over staining with the dissection microscope. On the fourth day it is important to incubate and bleach on a light box as this allows for any pigmentation in the samples or coloration from Bouin's solution to be removed, and enhances the probe signal for clear visualization.

Then after washing with SSC, carefully transfer the gut samples by inverting the basket into a new 24 well plate. While in wells, thoroughly rinse off the baskets using 70%ethanol. Then precede with imaging the samples.

It is critical to estimate the quality of the RNA probes before using them. The probes can be checked on a formaldehyde gel. If they appear as bright, discrete bands they are of good quality.

When staining some variation in distribution is normal among biological replicates as well as among the seven pairs of diverticula of individual guts. Therefore, it is best to stain at least five guts per condition to get an idea of average staining pattern. Overall success is best gaged by comparing the staining of sense and antisense samples for each condition.

Both must be developed for the same amount of time. The sense samples should have minimal to no purple color, while the antisense samples should display robust staining with minimal background. Critically, the tissues must be fully emersed in the stain for an even exposure.

It is important to avoid over development of the color reaction. This can result in a high amount of background staining, and the sense samples may begin to look like antisense samples. Another factor that affects results is how much the ticks are fed beforehand.

Ticks that are unfed, or fed for only 24 hours, are difficult to work with. Their guts are more easily damaged and do not stain well. Once mastered, this technique can be done in two and half to three days if it is performed properly.

While attempting this procedure, it is important to remember to keep all of the supplies listed in the table of reagents and materials ready before beginning your experiment. Thanks for watching, and good luck with your experiments.

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Journal (Genetics)

Journal (Genetics)

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

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