Non-radioactive in situ hybridization protocol applicable for Norway spruce and a range of plant species. Carre Etal. Hi, my name is AM GaN, and we are working at OBS University and the Swedish University of Agriculture Sciences in obs Sweden.
We have established an inset visitation protocol that works on several different plant species. Today we are going to show you some demanding steps. In this protocol, we are going to show you embedding sectioning and different steps in the actual inset visitation experiment.
Let's go to lab One, fixation and embedding Step nine to 11 in the protocol. Step nine, preheat Petri dishes and molds at 60 degrees centigrade. Step 10, heat a pair of tweezers and distribute tissues evenly in a dish.
Add more histo wax so that the tissues are covered. Step 11, let the wax harden at room temperature. Two sexually steps 12 to 19 in the protocol.
Turn on the hot plates and have melted histo wax available. Take out the samples from the cold room. Step 13, use a scalpel to carefully cut out a wax block of tissue.
Shape the piece to facilitate slicing. Step 14, heat the block cold and the heel fuse them together. Let the prep harden that four degrees centigrade.
Step 15, cut the wax block into six to eight micrometer thick sections. Using a microtone sections are linked in one long ribbon. Cut up a ribbon and move it to paper.
At this point, it is possible to inspect the individual sections under a stereo microscope To choose sections of interest. It'll be easy to perform an inspection with tissues or stained with eoin. Step 17 at RNA free Milli Q water onto the slides.
Cut the ribbon into smaller pieces and put them on the slides. Move the slides to the hot plate. Step 17, allow ribbons to flatten out on a 42 degrees centigrade plate for one to two minutes.
Drain off the water using Kim oil. Step 19, incubate the slides at 42 degrees centigrade overnight so that the tissues adhere to the slides four in situ hybridization 4.1 in-situ section. Pre-treatment steps 36 to 48 in the protocol.
Step 36 depa sections in histo layer. The slides are evenly spaced and placed in a rack to facilitate the movement between different containers, dehydrate sections. In an ethanol series, add acetic anhydrate while stirring vigorously.
Step 45, set up for acetic anhydride treatment 4.2 in situ hybridization. Steps 49 to 52 in a protocol. Step 51, aging of pro one plus slides.
The solution is viscous. Try to avoid bubbles. Incubate the slides in a humid chamber.
4.3 init. Post hybridization, steps 53 to 72 in the protocol. Step 62, incubate the slides with the burer block in a plastic container.
Step 63, add enough blocking solution just to cover the slides. The antibodies are added to the slides using aging. Aging only works with prob bon plus slides For slides of a similar design, Step 65, allow capillary forces to pull up the anti dig antibody solution.
This step is repeated. Step 70, as before sandwich and draw up the western blue solution. Notice that the Western blue solution is drawn up twice, just as we did for the anti dig antibody solution.
And step 65, step 71 incubate slides in darkness for one to five days. At room temperature, the monitoring of hybridization signals can be done in a stereo microscope. While the slides are still sandwiched, it should be possible to observe purple to brownish stains.
This is what the Results from actually in CW station experiment look like. You can see the signal here as purple or brownish staining.
