Name: Double Fluorescence in situ Hybridization in Fresh Brain Sections (Video) | JoVE Journal
Uploaded: 2010-08-14T17:00:00.000+00:00
Duration: 12 min 15 s
Description: Learn double fluorescence in situ hybridization for detecting two mRNA transcripts simultaneously in fresh brain sections at single cell resolution. This method reveals gene co-regulation in vertebrate neural circuits.

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5 minutes

Work supported by grants of NIH/NIDCD and the Schmitt Foundation to RP.

This protocol was developed and refined based on standard radioactive and non-radioactive in-situ hybridization methods, previously developed by us and others to detect one or two transcript species in brain tissue 1-7. The protocol described below has a total length of 2 or 3 days, depending on the number of procedural interruptions chosen by the end user. All steps detailed below are to be conducted at room temperature, with the exception of the riboprobe hybridization step and post-hybridization washes. The solutions and buffers required for all steps involved in this method can be found at the end of this protocol.
1. Tissue Preparation and Sectioning
<ol>
 <li>Decapitate and extract subject's brain rapidly, and place it into a plastic mold of adequate size.</li>
 <li>Cover brain with Tissue-Tek embedding medium and rapidly place the plastic mold in a dry-ice/alcohol bath for rapid freezing. Frozen tissue can be stored at -80°C until use.</li>
 <li>Using a cryostat, collect 2 or 3 brain sections per slide onto charged Superfrost Plus slides. Thickness of the sections should be 10-12 μm. Slides can be stored at -80°C until use.</li>
</ol>
2. Preparation of Sephadex G50 Columns for Probe Purification
"Sephadex columns can be purchased from commercial sources, however, we provide below a low-cost alternative for the generation of columns, which will be required for probe purification.
<ol>
 <li>Hydrate adequate amount of Sephadex G50 powder with RNase-free, DEPC-treated water (for example, 2 g of powder in 100 ml of DEPC-treated water), mix solution briefly and store at room temperature to precipitate excess Sephadex G50.</li>
 <li>Remove supernatant (upper layer of water) following Sephadex G50 precipitation.</li>
 <li>Repeat the process above 3- 5 times.</li>
 <li>After the final wash, re-suspend the Sephadex G50 solution in TE buffer (1:1 ratio), and store at 4°C until use.</li>
 <li>Place autoclaved glass wool into a sterile 1 ml syringe and compress it with the plunger to make a compact layer at the bottom, and then place the syringe into a 15 ml Falcon tube.</li>
 <li>Mix well the solution of Sephadex G50 in TE. Fill the syringe/column with this solution.</li>
 <li>Centrifuge the column for 30 sec at 1000 rpm.</li>
 <li>Repeat this procedure until the column is almost completely filled with Sephadex G50 beads.</li>
 <li>Apply 200 μl of column washing buffer to the column and centrifuge it for 2 min at 1000 rpm. Discard flow-through.</li>
 <li>Apply 200 μl of column blocking buffer to the column and spin it for 2 min at 1000 rpm. Repeat this step 4-5 times to equilibrate column. Columns can be stored at 4°C until use if sealed with Parafilm.</li>
</ol>
3. Labeling and Purification of Riboprobes
Below we detail the generation and purification of a single riboprobe. For dFISH, the preparation of each probe will involve the same methodology, except that one of the probes will be labeled with digoxigenin (DIG)-tagged UTP whereas the other with biotin-tagged UTP.
<ol>
 <li>Prepare a concentrated ( &gt; 150 ng/μl) and purified linearized cDNA solution of interest to generate either sense (control) or antisense riboprobes.</li>
 <li>In a 1.5 ml microfuge tube, add 0.5-1 μg of purified cDNA template, 2 μl of 5X probe labeling buffer, 1 μl of 10X DIG (or biotin)-labeling mix, 0.5 μl of RNasin and 1 μl of the appropriate RNA polymerase, and bring the final volume of the solution to 10 μl with RNAse-free (DEPC-treated) water.</li>
 <li>Incubate the solution in a water bath at 37°C for 2 hr.</li>
 <li>Add 1 μl of tRNA (stock; 20 μg/μl), and 39 μl of column blocking buffer to the solution.</li>
 <li>Prepare the Sephadex G50 column for probe purification. To this end, add 50 μl blocking buffer to the column and spin it for 10 sec at 1000 rpm. 
 Repeat this step 2-3 times to equilibrate the column (i.e., the full 50 μl applied to the column are recovered after cycle of centrifugation).</li>
 <li>Apply the probe solution to the Sephadex G50 column, position a new microfuge tube at the bottom of the column, and spin it for 3 min at 1000 rpm to obtain the purified 50 μl of riboprobe solution.</li>
 <li>Assess quality and yield of labeled riboprobe using either a standard formaldehyde-agarose RNA gel, or a spectrophotometer.</li>
</ol>
4. Post-fixation, Acetylation and Hybridization
<ol>
 <li>Remove sections from the -80°C freezer and allow for a room temperature to equilibrate. Subsequently, incubate sections in a cold, freshly-made 3% paraformaldehyde solution for 5 min.</li>
 <li>Briefly rinse sections in phosphate buffered saline (PBS) twice.</li>
 <li>Dehydrate sections through a standard alcohol series (70, 95, and 100%; 2 min each), and let them air-dry.</li>
 <li>Incubate sections in an acetylation solution for 10 min.</li>
 <li>Rinse sections 3 times in 2X SSPE.</li>
 <li>Dehydrate sections once again through the standard alcohol series described above, and allow them to air-dry.</li>
 <li>Prepare an adequate volume of hybridization solution and add both riboprobes to the solution (probe concentration: 1 ng/μl for each probe). Total volume of the hybridization solution is determined based on the number of sections to be hybridized (16 μl of hybridization solution per section).</li>
 <li>Apply adequate volume of the hybridization solution to tissue and coverslip slides ensuring that no air bubbles overlay the tissue.</li>
 <li>Place slides in a metal slide holder. Immerse holder in a mineral oil bath set at 65°C overnight. Ensure that coverslips are facing upward (i.e., the lateral aspect of the slide holder should be in contact with the bottom of the mineral oil container).</li>
</ol>
5. Post-Hybridization Washes
<ol>
 <li>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.</li>
 <li>Place slides in a 2X SSPE solution for 5-10 min. Coverslips should detach from slides while in solution.</li>
 <li>Transfer the slides to a new 2X SSPE solution, and keep them for 1 hr at room temperature.</li>
 <li>Transfer sections into a solution containing 2X SSPE plus 50% formamide. The temperature of this solution should match the temperature used in the overnight hybridization procedure. Keep slides in this solution for 1.5 hr.</li>
 <li>Transfer sections to a 0.1X SSPE solution pre-warmed to the same temperature of the hybridization step. Incubate sections in this solution for 30 min. Repeat this step for an additional 30 min.</li>
</ol>
6. Detection and Visualization of Riboprobes
<ol>
 <li>Transfer slides to TNT buffer to which 0.3% hydrogen peroxide has been added, for 10 min.</li>
 <li>Wash sections in TNT buffer, 3 times (10 min each).</li>
 <li>Using a DAKO pen, draw a well around the area containing the brain sections. Importantly, caution should be exercised to ensure that sections do not dry.</li>
 <li>Apply 150 μl of TNB buffer to each slide, and incubate sections in this solution for 30 min, in a humid chamber.</li>
 <li>Remove excess TNB solution by tilting slides.</li>
 <li>Apply 150 μl of TNB solution containing peroxidase-conjugated anti-DIG antibody, and store slides in a humid chamber for 2 hr. Antibody concentration should be determined individually for each transcript of interest prior to carrying out dFISH.</li>
 <li>Wash sections in TNT buffer; 3 times, 10 min each.</li>
 <li>Apply 150 μl of Alexa 594-conjugated tyramide working solution to each slide, and store them in a humid chamber for 1 hr. This solution should be prepared according to the manufacturer's directions.</li>
 <li>Wash sections in TNT buffer; 3 times, 10 min each.</li>
 <li>Incubate sections in TNT buffer to which 0.3% hydrogen peroxide has been added for 10- 30 min.</li>
 <li>Wash sections in TNT buffer; 3 times, 10 min each.</li>
 <li>Apply 150 μl of TNB buffer per slide, and keep them in a humid chamber for 30 min.</li>
 <li>Remove excess TNB solution by tilting slides.</li>
 <li>Add 150 μl of TNB solution containing peroxidase-conjugated anti-biotin antibody, and store slides in a humid chamber for 2 hr.</li>
 <li>Wash sections in TNT buffer; 3 times, 10 min each.</li>
 <li>Apply 150 μl of an Alexa 488-conjugated tyramide working solution to the slides and incubate sections for 1 hr. This solution should be prepared according to the manufacturer's recommendations.</li>
 <li>Wash sections in TNT buffer; 3 times, 10 min each.</li>
 <li>Add 150 μl of Hoechst solution (1:1000 in TNT buffer) to the sections and keep them in the humid chamber for 2 min.</li>
 <li>Wash the sections in TNT buffer; 3 times, 5 min each.</li>
 <li>Coverslip sections with a fluorescence-compatible mounting medium (e.g., Vectashield or ProLong antifade).</li>
</ol>
7. Representative Results
<img src="/files/ftp_upload/2102/2102fig1.jpg" alt="Fluorescence microscopy images showing protein expression patterns; green and red markers in cells." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. We show here representative dFISH results obtained in the zebra finch brain. Shown are photomicrographs obtained from the caudomedial nidopallium (NCM), the songbird analogue of the mammalian auditory cortex. Brain sections were hybridized with a biotinylated riboprobe directed against parvalbumin (A), a marker for a sub-population of inhibitory neurons, and a DIG-labeled riboprobe directed against the activity-dependent gene zenk (B), a reliable marker for song-driven neurons. C) Overlay of (A) and (B) shows a population of inhibitory neurons that are activated by auditory experience. Arrows and arrowheads indicate cells labeled exclusively with each of the two riboprobes, and asterisks show representative neurons co-expressing both transcripts of interest. Scale bar = 25 μm.

<ol><li>Jin, L., Lloyd, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+hybridization%3A+methods+and+applications%5BTitle%5D)+AND+%22J+Clin+Lab+Anal%22%5BJournal%5D)">In situ hybridization: methods and applications.</a> J Clin Lab Anal. 11, 2-9 (1997).</li>
<li>Stoler, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+hybridization%5BTitle%5D)+AND+%22Clin+Lab+Med%22%5BJournal%5D)">In situ hybridization.</a> Clin Lab Med. 10, 215-236 (1990).</li>
<li>Komminoth, P., Werner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Target+and+signal+amplification%3A+approaches+to+increase+the+sensitivity+of+in+situ+hybridization%5BTitle%5D)+AND+%22+Histochem+Cell+Biol%22%5BJournal%5D)">Target and signal amplification: approaches to increase the sensitivity of in situ hybridization.</a> Histochem Cell Biol. 108, 325-3233 (1997).</li>
<li>Kessler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+digoxigenin%3Aanti-digoxigenin+%28DIG%29+technology--a+survey+on+the+concept+and+realization+of+a+novel+bioanalytical+indicator+system%5BTitle%5D)+AND+%22Mol+Cell+Probes%22%5BJournal%5D)">The digoxigenin:anti-digoxigenin (DIG) technology--a survey on the concept and realization of a novel bioanalytical indicator system.</a> Mol Cell Probes. 5, 161-205 (1991).</li>
<li>Panoskaltsis-Mortari, A., Bucy, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+hybridization+with+digoxigenin-labeled+RNA+probes%3A+facts+and+artifacts%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts.</a> Biotechniques. 18, 300-307 (1995).</li>
<li>Qian, X., Lloyd, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+developments+in+signal+amplification+methods+for+in+situ+hybridization%5BTitle%5D)+AND+%22Diagn+Mol+Pathol%22%5BJournal%5D)">Recent developments in signal amplification methods for in situ hybridization.</a> Diagn Mol Pathol. 12, 1-13 (2003).</li>
<li>Mello, C. V., Jarvis, E. D., Denisenko, N., Rivas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+song-regulated+genes+in+the+brain+of+songbirds%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Isolation of song-regulated genes in the brain of songbirds.</a> Methods Mol Biol. 85, 205-217 (1997).</li>
<li>Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GABAergic+neurons+participate+in+the+brain%27s+response+to+birdsong+auditory+stimulation%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">GABAergic neurons participate in the brain's response to birdsong auditory stimulation.</a> Eur J Neurosci. 20, 1318-1330 (2004).</li>
<li>Velho, T. A., Pinaud, R., Rodrigues, P. V., Mello, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Co-induction+of+activity-dependent+genes+in+songbirds%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">Co-induction of activity-dependent genes in songbirds.</a> Eur J Neurosci. 22, 1667-1678 (2005).</li>
<li>Tremere, L. A., Jeong, J. K., Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estradiol+shapes+auditory+processing+in+the+adult+brain+by+regulating+inhibitory+transmission+and+plasticity-associated+gene+expression%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Estradiol shapes auditory processing in the adult brain by regulating inhibitory transmission and plasticity-associated gene expression.</a> J Neurosci. 29, 5949-5963 (2009).</li>
<li>Pinaud, R., Mello, C. V., Velho, T. A., Wynne, R. D., Tremere, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+two+mRNA+species+at+single-cell+resolution+by+double-fluorescence+in+situ+hybridization%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.</a> Nat Protoc. 3, 1370-1379 (2008).</li>
<li>Pinaud, R., Jeong, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Duplex+fluorescence+in+situ+hybridization+in+the+study+of+gene+co-regulation+in+the+vertebrate+brain.+Methods+Mol+Biol%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Duplex fluorescence in situ hybridization in the study of gene co-regulation in the vertebrate brain. Methods Mol Biol.</a> 611, 115-129 (2010).</li>
</ol>

No conflicts of interest declared.

We have used this protocol to study how the vertebrate brain is neurochemically and functionally organized, and to determine how behaviorally-relevant sensory stimuli impact the genomic machinery of neurons in the adult brain 8-10. We have successfully used this method in brain tissue from mice, rats and songbirds, but anticipate that this protocol will be easily adaptable to brain sections obtained from an array of vertebrate species and, perhaps, non-neural tissues. Key controls required to obtaining reliabl...

<table><tbody><tr><td>DEPC</td><td>VWR international</td><td>IC15090225</td><td>toxic substance</td></tr><tr><td>PCR purification kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Formaldehyde</td><td>VWR international</td><td>BDH0506-4LP</td><td>toxic substance</td></tr><tr><td>T3 RNA polymerase</td><td>Roche Group</td><td>11031163001</td><td></td></tr><tr><td>T7 RNA polymerase</td><td>Roche Group</td><td>10881767001</td><td></td></tr><tr><td>Tris-HCl (1 M, pH 7.5)</td><td>VWR international</td><td>IC816124</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; (1 M)</td><td>Sigma-Aldrich</td><td>449172</td><td></td></tr><tr><td>Spermidine (1 M)</td><td>Sigma-Aldrich</td><td>S0266</td><td></td></tr><tr><td>DIG RNA labeling mix</td><td>Roche Group</td><td>NC9380805</td><td></td></tr><tr><td>Biotin RNA labeling mix</td><td>Roche Group</td><td>NC9440104</td><td></td></tr><tr><td>RNasin</td><td>Promega Corp.</td><td>N2111</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>05491</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D5545</td><td></td></tr><tr><td>Sephadex G50</td><td>VWR international</td><td>95016-772</td><td></td></tr><tr><td>EDTA</td><td>VWR international</td><td>101384-758</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L4390</td><td></td></tr><tr><td>Transfer (t)RNA</td><td>Invitrogen</td><td>15401011</td><td></td></tr><tr><td>10X MOPS</td><td>VWR international</td><td>14221-398</td><td>toxic substance</td></tr><tr><td>Paraformaldehyde</td><td>VWR international</td><td>AAAL04737-36</td><td>toxic substance</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich</td><td>S3139</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>S3264</td><td></td></tr><tr><td>NaOH</td><td>VWR international</td><td>SX0600-1</td><td></td></tr><tr><td>Triethanolamine</td><td>VWR international</td><td>IC15216391</td><td></td></tr><tr><td>Acetic anhydride</td><td>VWR international</td><td>MK242002</td><td>toxic substance</td></tr><tr><td>SSPE</td><td>VWR international</td><td>82021-488</td><td></td></tr><tr><td>Formamide</td><td>VWR international</td><td>JT4028-1</td><td>toxic substance</td></tr><tr><td>Poly A</td><td>Invitrogen</td><td>POLYA.GF</td><td></td></tr><tr><td>Mineral oil</td><td>VWR international</td><td>IC15169491</td><td></td></tr><tr><td>Chloroform</td><td>VWR international</td><td>BDH1109</td><td>organic solvent</td></tr><tr><td>Deionized formamide</td><td>Sigma-Aldrich</td><td>F9037</td><td>toxic substance</td></tr><tr><td>Hydrogen peroxide</td><td>VWR international</td><td>VW36901</td><td>toxic substance</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Triton X-100</td><td>JT Baker</td><td>X198-05</td><td></td></tr><tr><td>Anti-DIG HRP</td><td>Roche Group</td><td>11093274910</td><td></td></tr><tr><td>Anti-biotin peroxidase</td><td>Vector Laboratories</td><td>SP3010</td><td></td></tr><tr><td>TSA Alexa Fluor 594</td><td>Invitrogen</td><td>T20935</td><td></td></tr><tr><td>TSA Alexa Fluor 488</td><td>Invitrogen</td><td>T20932</td><td></td></tr><tr><td>H&amp;#8211;chst</td><td>VWR international</td><td>200025-538</td><td></td></tr><tr><td>Vectashield</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>embedding mold</td><td>VWR international</td><td>15160-157</td><td></td></tr><tr><td>cryostat</td><td>Leica Microsystems</td><td>CM 1850</td><td></td></tr><tr><td>Superfrost plus slide</td><td>VWR international</td><td>89033-052</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Solutions&lt;/p&gt;&lt;ol&gt;&lt;li&gt;Column washing buffer: 10 mM Tris-HCl, 0.15 M NaCl, 0.05 mM EDTA, 50 &amp;#x03BC;g/&amp;#x03BC;l tRNA, 0.1% SDS in 50 ml DEPC-treated water.&lt;/li&gt;&lt;li&gt;Column blocking buffer: 10 mM Tris-HCl, 50 mM NaCl, 0.1 mM EDTA in 50 ml DEPC-treated water.&lt;/li&gt;&lt;li&gt;TNT buffer: 60 ml of 1 M Tris-HCl, 18 ml of 5 M NaCl and 1.8 ml of Triton X-100 in 600 ml of DEPC-treated water.&lt;/li&gt;&lt;li&gt;TNB buffer: 100 mM Tris-HCl, 8.3 &amp;#x03BC;g/&amp;#x03BC;l BSA, 0.15 M NaCl and 3% Triton X-100 in DEPC-treated water.&lt;/li&gt;&lt;li&gt;TE buffer: 10 mM Tris-HCl, pH 7.5 plus 1 mM EDTA, pH 7.5.&lt;/li&gt;&lt;li&gt;Acetylation solution: 2.7 ml of triethanolamine plus 0.5 ml of acetic anhydride in 200 ml of DEPC-treated water.&lt;/li&gt;&lt;li&gt;Hybridization solution: 50% formamide, 2X SSPE, 2 &amp;#x03BC;g/&amp;#x03BC;l tRNA, 1 &amp;#x03BC;g/&amp;#x03BC;l BSA and 1 &amp;#x03BC;g/&amp;#x03BC;l poly A in DEPC-treated water. It needs 16 l per section.&lt;/li&gt;&lt;/ol&gt;</td></tr></tbody></table>

double fluorescence in situ hybridization in fresh brain sections

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

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.

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Research

JoVE Journal

Neuroscience

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices.  By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal...

 Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

Source: Baleriola, J., et. al., Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization. J. Vis. Exp. (2015)This video demonstrates the detection of mRNA within cholinergic axons in fixed mouse hippocampal slices using fluorescent in situ hybridization and antibody staining. Fluorescence microscopy reveals the spatial localization of probe-tagged mRNAs and choline acetyltransferase in cholinergic neurons.

Immunostaining of Neurons in Mouse Brain Slices Following Fluorescence In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

Histological-Based Stainings Using Free-Floating Tissue Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

We present a detailed protocol for Epon post-embedding correlative light and electron microscopy using a fluorescent protein called mScarlet. This method can maintain the fluorescence and the ultrastructure simultaneously. This technique is amenable to a wide variety of biological applications.

Epon Post Embedding Correlative Light and Electron Microscopy

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

This study presents a simplified protocol for tissue processing involving decapitation, fixation, cryosectioning, fluorescence staining, immunostaining, and imaging, which can be extended to confocal and multiphoton imaging. The method maintains efficacy comparable to complex dissections, bypassing the need for advanced motor skills. Quantitative image analysis provides extensive investigative potential.

Direct Cryosectioning of Drosophila Heads for Enhanced Brain Fluorescence Staining and Immunostaining

Learn double fluorescence in situ hybridization for detecting two mRNA transcripts simultaneously in fresh brain sections at single cell resolution. This method reveals gene co-regulation in vertebrate neural circuits.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

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