The overall goal of the following experiment is to visualize mRNA expression patterns at the cellular level with high sensitivity and specificity. This is achieved by first taking formal and fixed and paraffin embedded tissue sections through a series of solutions to remove the wax perme the tissues and block positively charged minor groups in the tissues that can yield a background signal. Subsequently, A DIG labeled RNA probes specific for the gene of interest is infiltrated into the tissue sections and the slides are hybridized overnight.
Next, the slides are treated with RNAs. A to remove non-specifically bound probe from the sections and then incubated with anti DIG antibody. That allows the visualization of the hybridized probe by a color metric chemical reaction.
The results show dark blue signal detectable by light microscopy specifically in those cells within the tissue in which the gene of interest is expressed. The main advantage of this technique of our other expression analysis, such as R-T-P-C-R, microray or Next Generation Sequencing approaches is that the I Hybridization protocol shown here reveals the expression pattern of a gene of interest in its tissue context. These methods include many steps that are best learned by visual demonstrations.
We will show how to section and embed tissue as well as key steps in the Al Hybridization pro protocol that are difficult to master on your own. The method of embedding paraldehyde fixed tissues will vary depending on the type of tissue to be analyzed. The most important point here is to ensure that the samples are oriented correctly for sectioning later.
One method of embedding is to use plastic molds and rings. To begin this procedure, warm up the molds on a 60 degree Celsius warming plate and then pour molten paraffin into the molds. Next, use warmed forceps to transfer a tissue sample into each mold and orient it into the desired position.
Carefully move the mold from the plate to the bench and place the white ring onto the mold. Add additional mol and wax. Let the wax cool down and harden gradually prior to sectioning.
Warm the blocks to room temperature, then trim each block into a trapezoid shape, leaving about one millimeter of wax around the sample. Place the trimmed block onto the microtome such that the longer of the two parallel faces is at the bottom. Carefully bring the block forward to the blade and make sure that the surface of the block is parallel to the blade section.
As a thickness of eight to 10 microns. Align the wax ribbons on a non-stick surface, such as aluminum foil and select sections of interest under a dissection scope. Next, mark prob bon plus slides with a pencil.
Do not use pens on markers because they will be erased during the INI two hybridization protocol. Distribute one milliliter of clean milli Q water onto each slide. Carefully float sections of interest on the surface of the water at room temperature.
Ensure that the shiny side faces the water. Transfer the slide slowly onto a slide warmer. Set at 35 to 37 degrees Celsius.
This temperature range as opposed to the commonly used 42 degrees Celsius prevents the formation of bubbles under the sections. After five minutes, pour off the water carefully but in one smooth movement so that the ribbon is lowered down onto the slide. Let the slides dry for at least several hours or overnight at 37 degrees Celsius so that the tissue adheres to prepare tissue sections.
For in situ hybridization, they are first depa and rehydrated for depa finalization. Incubate the slides in two changes of histic clear solution for 10 minutes each. To rehydrate the tissue sections, pass the slides through a series of decreasing concentrations of ethanol for 30 seconds each.
During the protease treatment, which is not shown in this video, prepare a glass dish with 0.1 molar triathlon amine solution and set this up on a stir plate in a fume hood. Immediately before placing the metal slide rack into the solution, add 1.25 milliliters of acetic anhydride into the triathlon amine, buffer and stairwell. In addition, set up the clamping system and stand for holding the slide rack.
Reduce the speed of the Starr clamp the slide rack onto the stand and lower the rack into the solution, keeping it elevated above the stir bar. Continue to stir slowly for 10 minutes after being rinsed in PBS and dehydrated again through an ethanol series. The tissue sections are ready for hybridization.
Place the slides on a clean surface and let the air dry completely for five to 10 minutes. Prepare the probe hybridization buffer mixture by adding the denatured probe to 80 microliters of hybridization buffer. For each slide, mix well by pipetting plus, avoid making bubbles.
Pipette the probe hybridization buffer. Mix onto the right edge of the slide. Gradually lower a cover slip onto the slide, making sure that the hybridization solution covers all sections without any bubbles.
Tap the cover snip lightly with your finger where bubbles form to displace them from all tissue sections. Do not pull the cover snip off as this will damage the sections. Place the slides in a humidity chamber containing watman paper moistened with UE water and largely covered with param.
Seal the box tightly incubate the slides at the desired hybridization temperature, typically 50 to 55 degrees Celsius for 16 to 20 hours. At the completion of the hybridization protocol, carefully remove the cover slips from the slides. The cover slip may simply fall off when the slide is placed upright, but if not, do not pull off the cover slip as this will damage the sections.
Instead, immerse the slide in warm 0.2 times SSC for one or two minutes to wash the cover slip off after removing cover slips, place the slides in a rack and wash several times in 0.2 times SSC at 55 degrees Celsius. After RNAs a treatment transfer the slides from the rack onto the bottom of a flat plastic box and cover with a minimal volume of blocking solution. Block the slides for 45 minutes of room temperature on a slowly shaking platform.
Next, apply a minimal volume of antibody solution directly onto each slide. Incubate the slides in the dark for two to three hours at room temperature. When the incubation is complete, transfer the slides to a clean flat box and wash the slides four times with a minimal volume of washing buffer on a shaking platform at room temperature.
Rinse the slides once in TBS and twice in TN buffer offer to apply freshly prepared staining solution to the slides. First, pour the staining solution into a small plastic weighing dish. Then sandwich two slides together with the sections facing each other.
Dip the sandwich into the solution, allowing capillary action to pull up the solution. Drain the slides on a kim wipe. Fill the slides with staining solution.
Again, the slides may need to be tapped as the solution flows in to avoid bubbles, place the slides in a plastic box and store room temperature in the dark. Representative results attained with different probes and arabidopsis tissues are shown here. The purple signal provides a direct visualization at cellular resolution of the expression pattern of the gene of interest.
Panel A shows the localization of shoot Meli one transcripts in the vegetative shoot apex. Note the presence of purple blue signal in the indeterminate of the meristem and the lack of signal in the surrounding leaves. Panels B 2D are sections of arabidopsis torpedo stage embryos where B shows covata three expression in the few embryonic stem cells.
C shows a T ML one expression in the epidermal layer, and D shows the lack of signal in sections probed with a random negative control RNA. These next images showed results obtained with different probes on maze tissues. Panel E shows the localization of the STM Humalog knotted one in the developing maze embryo.
Note the strong signal specifically in the embryonic mes stem and root longitudinal sections through the vegetative chute apex show that ARF three A is expressed on the axial bottom leaf surface in panel F and the A T ML one homolog OCL four is expressed specifically in the epidermis. In panel G.As expected, no hybridization signal was observed for the negative control probe in panel H.The expected results of the different checkpoints during the in vitro transcription of probes are shown here. The in situ hybridization probes are checked on a standard ARAZ gel after in vitro transcription after DNA's treatment and subsequent purification of the in vitro transcription reaction After carbonate hydrolysis and when ready for use for probes over 250 base pairs in length such as probe one, the carbonate hydrolysis will yield a range of smaller probe fragments.
This figure shows the results of color and metric qualification of probes by dot blot analysis dilutions ranging from 10 to the minus one to 10 to the minus five of a 100 nanogram per microliter control probe and of three, newly labeled DIG labeled probes are spotted on a transfer membrane incubated with anti DIG antibody and assay. The analysis suggests an estimated concentration of 100 nanograms per microliter for probe two 10 nanograms per microliter for probe three and one nanogram per microliter for probe four, indicating that probe four is unlikely to yield a good inea to hybridization signal. Finally, these longitudinal sections through the vegetative chute apex from Mace provide a comparison between a non-specific background signal and a well-working probe panel.
A shows a non-specific background signal while panel B shows a specific in situ two hybridization signal for the OC five probe in the outer cell layer. After watching this, you should have a good sense of how to perform many of the tricky step in the init hybridization protocols such that you can determine the precise pressure temporal expression patterns of your favorite teens.
