The overall goal of this procedure is to detect RNA transcripts in place within cells and tissues. This method can help answer key questions in the cell and developmental biology fields such as where inside a cell, a tissue, or an embryo an RNA is located. The main advantage of this technique is that it detects RNA spatial distributions with both high levels of resolution and sensitivity and at a low cost compared to other methods.

We initially developed this protocol to study the subcellular distributions for as many Drosophila RNAs as possible. This was in order to determine the extent and diversity of subcellular distributions. We started the project working with embryo, but later adapted and optimized the protocol so we could work in high throughput with tissues dissected from larvae and flies.

The most difficult aspect of the procedure is the number of steps and the consequential potential for error as well as the need to ensure the absence of RNAs during probe production. To facilitate throughput and cost effectiveness, the procedures demonstrated in this video utilize 96-well microtiter plates for all steps. It is also easy to adapt this method to smaller sample members by cutting the plates into eight or 12-well strips.

Start by diluting 10 microliters of an overnight bacterial culture in 90 microliters of autoclaved double distilled water in each well of a new 96-well PCR plate. Denature the DNA at 95 degrees Celsius in a thermocycler for five minutes. Immediately place the plate on ice for at least five minutes.

Set up the PCR reactions in a new 96-well PCR plate. Use the appropriate universal primers and aliquot 48 microliters of the PCR master mix into each well. Add two microliters of each denatured plasmid DNA template per well.

Perform the amplification in a 96-well PCR machine. When the amplification is complete, precipitate the PCR product with ethanol sodium acetate mix. Store the samples for 30 minutes at minus 80 degrees Celsius.

Centrifuge the 96-well plate at 2, 250 times g at four degrees Celsius for 45 to 60 minutes. Using eight-channel vacuum manifold pipette which has a structure that prevents tips from going to the bottom of wells in sucking up the pellets, carefully remove the supernatant. Wash once with 160 microliters of cold 70%ethanol.

Centrifuge at 2, 250 times g at four degrees Celsius for 10 minutes. Gently invert the 96-well plate on a paper towel to remove the last drops of liquid in each well and air dry the DNA pellets at room temperature for one hour. Resuspend the precipitated DNA in 25 microliters of RNase-free water.

Check the size and yield of the PCR products by agarose gel electrophoresis. Set up the in vitro transcription reactions by adding the appropriate components to each well in a new 96-well PCR plate. Add 7.5 microliters of DNA template to each corresponding well.

Cover the plate with sealing tape. Incubate the plate at 37 degrees Celsius for 3-1/2 to four hours. Next, add to each well a master mix comprised of DEPC-treated double distilled water, 100%ethanol, and three molar sodium acetate.

Store at minus 80 degrees Celsius for 30 minutes. Centrifuge the plate and wash the wells as previously shown. Resuspend the precipitated probe in 25 microliters of DEPC-treated double distilled water.

Newly-synthesized antisense RNA probes were run on a 1%agarose gel to check for integrity and yield. Based on the intensity of the band, the yield can be characterized as low yield, typical yield, or high yield. After using five microliters of each probe for agarose gel electrophoresis, mix the remaining 20 microliters with 100 microliters of hybridization solution and store at minus 80 degrees Celsius until needed.

To start this procedure, place 20 to 30 larvae in a Petri dish containing cold PBS in fix one solution and chill on ice for two minutes to reduce larval motility. Dissect the larvae under the dissecting scope. Use a pair of sharp forceps to carefully open the anterior and squeeze the tissues from posterior to anterior.

After a maximum of 15 minutes, transfer the dissected tissues to a 1.5 milliliter tube for fixation and place on ice. Remove PBS and add 800 microliters of fix one solution. Incubate on a bench top mixer for 30 minutes alongside other previously collected in fixing tissues.

Rinse the tissues once with 800 microliters of PBTT and keep the tube on ice while additional tissues are being collected and fixed. Pool all dissected tissues into a single 15 milliliter plastic tube containing mesh in the tube lid. Aspirate excess liquid through the mesh in the lid.

Wash with 10 milliliters of PBTT and rinse with 10 milliliters of PBS. To quench endogenous horse radish peroxidase activity, add five milliliters of 0.3%hydrogen peroxide in PBS and incubate at room temperature for 15 minutes. Wash twice with 10 milliliters of PBTT.

Permeabilize the tissues by adding 10 milliliters of pre-chilled 80%acetone and incubating at minus 20 degrees Celsius for 10 minutes. Wash twice with 10 milliliters of PBTT to rehydrate the tissues. After washing the tissues as described in the text protocol, store in hybridization solution at minus 20 degrees Celsius.

If not used by the next day, the filter assembly should be replaced with a regular cap. Aliquot about 20 microliters per well of tissues into a 96-well PCR plate on ice. Using an eight-channel manifold pipette, remove the hybridization solution from the tissues.

Add 100 microliters of freshly denatured hybridization solution to each well and cover with sealing tape. Pre-hybridize the tissues at 56 degrees Celsius using a dry bath heating unit containing metal beads for a minimum of 2-1/2 to three hours. Denature 100 microliters of the prepared probe in a 96-well PCR plate at 80 degrees Celsius for five minutes.

Immediately cool on ice for five minutes. Remove the pre-hybridization solution from the tissues and add denatured gene-specific probes to each sample. Cover with aluminum sealing tape and hybridize at 56 degrees Celsius under metal beads in a dry bath heating unit for 16 to 18 hours overnight.

Prior to starting the probe detection procedure, pre-warm all the required solutions in the 56 degree Celsius heating unit. Carefully transfer hybridized tissues into a 96-well plate that fits a multi-well plate vacuum manifold. The wells of these plates have membrane bottoms that allow removal of contents under vacuum.

Remove the probes from the 96-well plate and wash the samples with mixtures of hybridization solution and PBTT per the steps indicated in this table in the 56 degree Celsius heating unit. After the third wash with PBTT, remove the 96-well plate from the heating unit, remove the PBTT solution, and block the tissues with PBTTB on a bench top mixer at room temperature for 20 minutes. Add 100 microliters of antibody solution per well and incubate for two hours on the bench top sample mixer.

After rinsing with PBTTB as described in the text protocol, add 100 microliters of Streptavidin horse radish peroxidase solution per well and incubate at room temperature for 1-1/2 hours. Wash twice with PBTTB for five minutes per wash. After this, incubate the tissues with DAPI solution as described in the text protocol.

Start this procedure by incubating the samples with Tyramide solution and incubate the samples for two hours on the bench top mixer in the dark. Remove Tyramide solution from tissues once the incubation is done. After washing with PBT and PBS as described in the text protocol, add 150 microliters of antifade mounting media per well.

Keep samples at four degrees Celsius overnight to allow tissues to sink to the bottom of the well or tube. On the following day, mount the samples on a microscope slide and seal with a coverslip. Lastly, image the samples using a fluorescence microscope.

Shown are examples of results obtained using this procedure for early Drosophila embryos and late Drosophila embryos with signals in amnioserosa, muscles, and the central nervous system. The name of the gene to which the probe hybridized is displayed in each panel. Next are examples from third instar larval tissues, malpighian tubules, midgut, salivary gland, and muscle.

Lastly, representative images from adult ovary and adult testes are shown. Once mastered and if performed efficiently, this technique can be done in as little as two days. While attempting this procedure, it's important to remember to work on an RNase-free bench with RNase-free tips, tubes, plates, and solutions and to use gloves.

If you're working with a large number of samples or probes, don't rush through the procedure. And it's also a good idea to come up with tricks so that you don't forget where you are. When you're following this procedure, you can also combine it with other methods like detecting multiple probes, probes with proteins or cellular markers.

With these you can answer additional questions such as relative expression patterns, cell and subcellular compartment identities, and effects in different genetic backgrounds or diseases. After watching this video, you should have a good understanding of how to perform most types of in situ hybridization in any type of tissue or embryo. Don't forget that working with reagents like formaldehyde, hydrogen peroxide, and formamide can all be extremely hazardous and precautions such as proper handling and disposal should always be taken while performing the procedure.

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