Double Fluorescence in situ Hybridization in Fresh Brain Sections

Hi, I'm JA postdoctoral research associate at AL helping laboratory at the Universal of Rochester. We will show you the Process Institute hybrid digestion in fresh frozen brain sections to detect two MRN transcripts simultaneously at single cell resolution. This method can help ask you questions in the neuroscience field, such as practice relating to the anatomical functional molecular and neurochemical organization of a tablet brain circuit.

Though this method can provide insights into gene coagulation in neurons of the AV brain. It can also be applied to other systems such as rodents, as well as a variety of the neural tissues. To begin this procedure, hydrate an adequate amount of SDX G 50 powder with RNAs through water.

Mix the solution briefly and store room temperature for two to five minutes. This step separates the SDX beads from the dextrose found in commercial sedex powder following the Sedex G 50 precipitation. Remove the sennet and repeat the process three to five times after the final wash reus suspend the SX G 50 solution in TE buffer at a one-to-one buffer to bead volume ratio and store at four degrees Celsius until use.

Next, prepare the columns. Place autoclave glass wool into a sterile one milliliter syringe and compress it with the plunger to make a compact layer at the bottom. Then place the syringe in a 15 milliliter falcon tube.

Fill the columns with Sedex G 50 solution with centrifugation at 1000 rotations per minute for 30 seconds using a Beckman centrifuge fitted with a C 0 6 5 0 fixed angled rotor. After centrifugation. Wash each column by applying 200 microliters of column washing buffer and centrifuging for two minutes at 1000 rotations per minute.

As before. Finally apply 200 microliters of column blocking buffer to each column and spin for two minutes at 1000 rotations per minute. Repeat this step four to five times to equilibrate the column.

Store the prepared columns at four degrees Celsius until the RIBA probe is labeled and ready for purification for the double fluorescence in C two hybridization or D FISH procedure shown here, two RNA probes are prepared, one labeled with doxygen in or DIG insured and the other with biotin. Start labeling the probes by preparing the ribo probe labeling solutions and incubating the labeling reactions in a water bath at 37 degrees Celsius for two hours. At the end of the two hour incubation, add one microliter of 20 micrograms per microliter TRNA and 39 microliters of column blocking buffer to each labeling reaction.

Then prepare the EDEX G 50 columns for probe purification by adding 50 microliters of blocking buffer to each column and spinning for three minutes at 2000 rotations per minute. Repeat this step two to three times to equilibrate the columns. When the columns are ready, place a new EEND orph tube at the bottom of the column.

Apply the probe solution to the Sedex G 50 column and spin for three minutes at 2000 rotations per minute. To obtain the purified 50 microliters of Ribo probe solution, assess the quality and yield of the labeled and purified ribo probe. Before proceeding to hybridization using a cryostat, collect two or three brain sections onto charge super frost plus slides.

The thickness of the section should be 10 to 12 microns. The slides can be stored at minus 80 degrees Celsius until use. Incubate the sections in cold freshly made 3%para formaldehyde solution for five minutes.

Then briefly rinse the sections twice in PBS. Next, dehydrate the sections through a standard alcohol series of 70, 95 and 100%ethanol solutions for two minutes in each solution. After dehydrating the sections, incubate them in freshly prepared acetylation solution for 10 minutes.

Then rinse the sections three times in two times SSPE dehydrate sections following acetylation once again through the standard alcohol series as just shown. Now start the hybridization with the ribo probe. Apply an adequate volume of the hybridization solution to the tissue cover the slides with cover slips and place slides in a metal slide holder.

Immerse the holder in a mineral oil bath set at 65 degrees Celsius overnight. The following day. Carefully remove the slide holder from the mineral oil bath and briefly rinse it in chloroform to remove excess oil from the slides.

Place the slides in a two times SSPE solution for five to 10 minutes in order to remove the cover slip. After removing the cover slip, transfer the slides to new two times SSPE solution and keep them for one hour at room temperature at the beginning of this incubation step prewarm solution of two times SSPE plus 50%form aide to the same temperature as in the hybridization. Step at the end of the one hour incubation.

Transfer the slides into the pre-war two times SSPE for maide solution and incubate for 1.5 hours. At the beginning of this incubation step prewarm a solution of 0.1 times SSPE to the same temperature as in the hybridization step. Finish the post hybridization washes by transferring the slides to the PREWARM 0.1 times SSPE solution and incubate for 30 minutes.

Repeat this wash once. The brain sections are now ready for detection and visualization of the hybridized ribo probes had 0.3%hydrogen peroxide to TNT buffer and incubate the slides in this solution for 10 minutes. Then wash the slides in TNT buffer three times for 10 minutes each time using a deco pen.

Draw a well around the area containing the brain sections. Apply 150 microliters of TNB buffer to each slide and incubate the sections in this solution for 30 minutes. Make sure the sections do not dry out during this step.

Next, apply 150 microliters of TT NB solution containing peroxidase conjugated anti DIG antibody and store the slides in a humid chamber for two hours. At the end of the two hour incubation, wash the sections in TNT buffer three times for 10 minutes. Each time apply 150 microliters of Alexa 5 9 4 conjugated T IDE working solution to each slide and store the slides in a humid chamber for one hour.

At the end of one hour, wash the sections in TNT buffer three times for 10 minutes. Each time incubate the sections in 0.3%hydrogen peroxide in TNT buffer solution for 10 to 30 minutes. The incubation time in the hydrogen peroxide solution depends on mRNA abundance and should be tested prior to D fish.

Then wish the sections in TNT buffer three times for 10 minutes each time. Apply 150 microliters of TMB buffer per slide and keep the slides in a humid chamber for 30 minutes. Remove excess TMB solution by tilting the slides.

Add 150 microliters of TMB solution containing peroxidase conjugated anin antibody, and store slides in a humid chamber for two hours. Then wash the sections in TNT buffer three times for 10 minutes. Each time apply 150 mic releases of an Alexa 4 88 conjugated mite working solution to the slides and incubate sections for one hour.

Then wash the sections in TNT buffer three times for 10 minutes each time. Now that both ribo probes are labeled by antibodies, incubate the sections in 150 microliters of host solution for two minutes. Then wash the sections in TNT buffer three times for five minutes each time, add a fluorescence compatible mounting medium such as vector shield or prolonged antifa to the sections cover with a cover slip and observe under a fluorescent microscope.

These representative photo micrographs are from a D fish Experiment with zebra finch brain sections, specifically from the NCM region, the songbird analog of the mammalian auditory cortex. Brain sections were hybridized with two probes, a biotinylated ribo probe directed against par albumin, a marker for a subpopulation of inhibitory neurons and a DIG labeled ribo probe directed against the activity dependent gene zinc, A reliable marker for song driven neurons. An overlay of the two images shows a cell population of inhibitory neurons that are activated by auditory experience.

Arrowhead indicate cells labeled exclusively with the ribo probe for par. Albumin arrows indicate cells labeled exclusively with the RIBA probe for zinc representative neurons Coex expressing both transcripts of interests are marked with asterisk. While attempting this procedure, it is important to remember to maintain your bench clean and use evidence free solutions, plastics and glassware.

After watching this video, you should have a good understanding of how to simultaneously detect two MA species, a single cell resolution in freshly frozen brain sections to study gene co-regulation in the we rate brain. Thank you for watching the video. Good luck with your experiments.