This is an ceto protocol for butterfly wings at the pupil stage. So we start by collecting pre pupa from the cage, setting them on a tray, and then photographing their exact PPU patient times by using time lapse photography. This allows us to do the stainings at precise times after pupation occurs.
This is important because different mRNAs for different proteins are expressed at different times after pation. Here we're dissecting the pupil wings in a Petri dish with a silicone base, and the important part is to remove the wings and put them in the fix solution so that they are very flat. If they're not flat at this stage, they will never become flat again.
After the right fixation time, we remove the fixed buffer and we wash five times. With PBT, the proteinase case step is rather critical, and it's used to per Alize the tissues for the probe to it should be stopped at the right time with these rinses and washes. The salmon sperm works as a cheap source of DNA to prevent the probe from binding everywhere.
The sperm is one of the components of the pre hybridization buffer. So here we are adding the sperm to the pre hybridization buffer, and then there's going to be a solution made, which is this 1 50 50 P-B-T-P-H-B half, half of PBT and pre hybridization buffer. This solution is used as an intermediate solution to bring the wings gently from the PBT solution where they're baited in to the pre hybridization solution.
And so there's an intermediate step where the wings are going to be sitting initially in this 50 50 solution. Here, the wings are being incubated in PHP for 10 minutes, and then again in PHP for one hour at 55 degrees. In the meantime, we're going to be making the hybridization buffer, which is just pre hybridization buffer mixed with glycogen.
So here we're adding glycogen to PHV to make a small amount of hybridization buffer to this buffer, we're going to be adding the denatured probe. So we are warming up a heating block and we are spinning down the probe that has been stored at minus 20 and move just the right amount of probe to a YouTube to be denatured at 80 degrees for about five minutes. Here we are denaturing two probes simultaneously, so we add a hybridization buffer to the probe, and then we add a hundred microliters of probe mix to each well.
To finalize this step, we'll just cover all the wells with perfil because the wings will be incubated for the next two days at 55 degrees, and the parfum prevents evaporation After two days of incubation, the probe is removed by washing the wings with PHP and the last wash stays on the wings for another 12 to 24 hours. Finally, the wings are washed through a series of steps to bring them back to PBT solution. So again, using the 50 50 mix solution in between.
At this stage, the antibody block solution is added to the wings and the anti dig antibody conjugated to alkaline phosphatase is added to PBT to make a concentration of about one to 2000. About 300 microliters of this antibody solution will be added to each well, and the wings will then be set in the fridge incubating in antibody overnight. The next day, the antibody is removed by doing a series of washes with PBT, and then the wings are going to be rinsed twice in the detection buffer at room temperature.
Finally, one mill of developing solution is added to each well and the wings rapidly covered with the aluminum foil. After about 10 minutes, we check and the wings are starting to look purple, but they're still a little light. So they're covered again, and some minutes later we check again and they've become a little darker.
Notice that wings that were incubated in different probes seemed to darken its slightly different rates, so we decide to cover them again, and finally, 20 minutes later, we decide to stop the reaction with the development stop buffer. The wings are now ready to be mounted on a slide, so we add a little droplet of mounting medium to a labeled slide and move the wings to the slide by just picking them up with tweezers and setting them on the droplet. Move away any air bubbles and gently straighten the wing and cover it with a cover slip.
In this experiment, we use the probe that targets in grail gene transcripts, and we see them localized in the center of the future IPO pattern of the butterfly by cyclist anana during the early stages of pupil wing development.
