Fluorescent ISH Using DIGLabeled Probes

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3.1.1. Tissue Preparation

For this ISH method, the whole procedure is carried out on free-floating sections obtained after perfusion fixation, postfixation, sucrose embedding, and cryosectioning of the brain (see Note 12).

3.1.1.1. Perfusion

1. Anesthetize and perfuse with heparin-PBS followed by 4% PFA using a regular perfusion protocol.

2. Decapitate and remove brain carefully.

3.1.1.2. Sucrose Embedding

1. Drain PFA from the vessel containing the brain and fill it with 20% sucrose.

2. Store at 4°C for 48 h; this treatment will cryoprotect the sample.

3. Remove the brain from the vessel, blot the excess sucrose solution with a paper towel, wrap the brain in tape-labeled aluminum foil, and freeze the brain at -80°C until ready for sectioning. Brains can be stored at -80°C for several months.

3.1.2. In Vitro Transcription of DIG-Labeled Riboprobe

3.1.2.1. DNA Template

As indicated in Fig. 1, two methods can be used to obtain DNA templates. The first method involves the synthesis of relatively large amounts of an in vitro transcription product from a plasmid vector containing an insert of the gene of interest (see Note 13). This is done by transformation of competent bacteria, followed by bacterial culturing and purification of plasmid DNA by mini-, midi-, or maxi-preparation. For general guidelines on how to perform these procedures refer to ref. 6 and to the QIAGEN Plasmid Purification Hand-

Fig. 1. Generation of DNA templates for riboprobe synthesis. (A) Schematic drawing of a generic transcription plasmid vector where a cDNA fragment of interest has been cloned (clock gene X cDNA insert, framed by white rectangle). The large white arrow indicates the original (in vivo) direction of transcription of the gene. In order to use the cDNA insert as a template for transcription of RNA the plasmid must be linearized with the appropriate restriction enzyme (specific sequence for the enzyme in black, flanking the insert) and transcribed with the appropriate RNA polymerase (T7 or T3, specific promoter sequences indicated in gray). In this case, digestion with EcoRI and transcription with T7 RNA polymerase and labeled nucleotides (*) will yield a sense RNA probe. Instead, digestion with NotI and transcription with T3 RNA polymerase will result in synthesis of an antisense RNA probe, which should hybridize and label the gene's mRNA in the tissue (continued on next page).

Fig. 1. Generation of DNA templates for riboprobe synthesis. (A) Schematic drawing of a generic transcription plasmid vector where a cDNA fragment of interest has been cloned (clock gene X cDNA insert, framed by white rectangle). The large white arrow indicates the original (in vivo) direction of transcription of the gene. In order to use the cDNA insert as a template for transcription of RNA the plasmid must be linearized with the appropriate restriction enzyme (specific sequence for the enzyme in black, flanking the insert) and transcribed with the appropriate RNA polymerase (T7 or T3, specific promoter sequences indicated in gray). In this case, digestion with EcoRI and transcription with T7 RNA polymerase and labeled nucleotides (*) will yield a sense RNA probe. Instead, digestion with NotI and transcription with T3 RNA polymerase will result in synthesis of an antisense RNA probe, which should hybridize and label the gene's mRNA in the tissue (continued on next page).

book (Qiagen). After purification, the plasmid should be resuspended in TE buffer or in DEPC-treated water at a concentration of 1 ^g/^L and stored at -20 or -80°C. Linearization of the plasmid with the appropriate restriction enzyme (see Fig. 1A and Note 14) can be done according to the enzyme manufacturer's instructions. Linearization should be confirmed by running a sample of the reaction product and the original nonlinearized plasmid in a 1% agarose gel. The nonlinearized plasmid should run slightly faster. Incomplete digests, which must be avoided, yield two bands. After the linearization reaction is completed,

Linearization Dna

Fig. 1. (continued) (B) Schematic drawing of a double strand of DNA that contains a cDNA fragment of interest but no RNA polymerase promoter sequences flanking it. In this case the DNA template with the RNA polymerase promoter sequences is generated by polymerase chain reaction amplification using primers that flank the region of interest and have the RNA polymerase promoter sequences as overhangs (free "tails" in gray). Remaining symbols as in (A). Although this method to generate the template is different, in this example T7 and T3 RNA polymerization of the template will also yield antisense and sense RNA, respectively. Notice that with both methods, the labeled riboprobe will carry some labeled nucleotides that do not correspond to the DNA insert (outside white rectangles), but these nucleotides will not impair hybridization of the riboprobe to the specific mRNA.

Fig. 1. (continued) (B) Schematic drawing of a double strand of DNA that contains a cDNA fragment of interest but no RNA polymerase promoter sequences flanking it. In this case the DNA template with the RNA polymerase promoter sequences is generated by polymerase chain reaction amplification using primers that flank the region of interest and have the RNA polymerase promoter sequences as overhangs (free "tails" in gray). Remaining symbols as in (A). Although this method to generate the template is different, in this example T7 and T3 RNA polymerization of the template will also yield antisense and sense RNA, respectively. Notice that with both methods, the labeled riboprobe will carry some labeled nucleotides that do not correspond to the DNA insert (outside white rectangles), but these nucleotides will not impair hybridization of the riboprobe to the specific mRNA.

the linearized plasmid DNA should be purified by phenol/chloroform purification and ethanol precipitation, or alternatively by using Quick Spin Columns (Roche). Purified DNA can be resuspended in TE buffer or DEPC-treated water and stored for at least 1 yr at or -80°C. See ref. 6 for basic techniques.

The second method for obtaining DNA templates is by polymerase chain reaction (PCR) amplification of the DNA fragment of interest, using primers with RNA polymerase promoter sequences as overhangs (see Fig. 1B and Note 15). A sample of the PCR product should be run in a 1% agarose gel to confirm that a single band of the expected size is obtained. The amplified DNA can be purified and stored by the same methods used after linearization of plasmids.

3.1.2.2. Riboprobe Synthesis

Labeling of RNA probes for fluorescent detection of specific mRNA is based on the tagging of specific RNA bases with DIG, and the subsequent fluores cent immunolabeling of the DIG molecules. The DIG-labeling kit uses DIG-labeled UTP as a source of uracils for the riboprobe.

1. Using 1 g of template DNA perform the labeling reaction according to the instructions of the DIG RNA labeling kit (Roche), which contains all the necessary components (see Note 16). Perform the optional DNase I treatment.

2. Purify the probe by ethanol precipitation. Do not use phenol/chloroform purification because the DIG-labeled RNA will partition into the organic phase.

3. Resuspend the riboprobe in 40 pL of DEPC water and aliquot into smaller volumes, store at -80°C. Under this condition probes are stable for at least 1 yr if not repeatedly frozen and thawed.

4. To quantify the probe, make a dilution series of the probe and of the labeled control RNA as indicated in the kit instructions. Apply 1 pL spot for each dilution of control and synthesized riboprobes on a nylon membrane. Air-dry the membrane and crosslink the RNA either by UV or baking, according to the manufacturer's instructions.

5. Incubate the dot blot for 1 h in blocking solution, then overnight in 1:500 dilution of peroxidase-conjugated anti-DIG antibody in buffer 1 (see Subheading 3.1.5. for details). Peroxidase staining is done with diaminobenzidine following the manufacturer's instructions.

Figure 2 shows an example of a dot blot where the labeled control RNA and two probes were applied after a 1:2 dilution series. Using the known concentration of the control RNA, the concentration of the DIG-labeled probe can be roughly estimated. When using a sense probe as control for ISH, it is important to use a similar concentration (based on the intensity of DIG staining in the dot blot) to that of the antisense probe (see Note 17).

3.1.3. Sectioning, Prehybridization, and Hybridization

3.1.3.1. Sectioning

Tissue should be cut on the same day that the ISH is started. All buffers for prehybridization and hybridization should therefore be prepared before brain sectioning starts. Sections can be cut in either a freezing microtome or a cryostat. In both cases they will be kept free-floating throughout the procedure.

1. Remove the brains from -80°C and place them in dry ice.

2. Cut the brains into 40- to 50-pm sections (see Note 18).

3. As you cut, transfer the sections to netwells placed in 12-well cell culture microplates containing chilled 2X SSC. Sections are kept at 4°C until the prehybridization is started. Leave every other well without a netwell for each set of sections.

3.1.3.2. Prehybridization

Unless indicated, the whole procedure is done by switching the netwells back and forward, after rinsing the well with 2X SSC and replacing with the

Fig. 2. Dot blot of digoxigenin (DIG)-labeled RNA. The membrane shows a blot of 1 ^L of a 1:2 dilution series of DIG riboprobes for rPerl sense and antisense, for mBMALl antisense, and for the Roche RNA labeled control. After staining, the RNA concentration of each riboprobe solution can be roughly estimated based on the concentration of the labeled control RNA. Notice that the labeling efficiency is higher for rPerl antisense than for rPerl sense probe. If this sense probe is used as a control for antisense labeling by in situ hybridization, the same final concentration of sense and antisense probe, based on this dot blot estimation, should be used in the hybridization reaction.

Fig. 2. Dot blot of digoxigenin (DIG)-labeled RNA. The membrane shows a blot of 1 ^L of a 1:2 dilution series of DIG riboprobes for rPerl sense and antisense, for mBMALl antisense, and for the Roche RNA labeled control. After staining, the RNA concentration of each riboprobe solution can be roughly estimated based on the concentration of the labeled control RNA. Notice that the labeling efficiency is higher for rPerl antisense than for rPerl sense probe. If this sense probe is used as a control for antisense labeling by in situ hybridization, the same final concentration of sense and antisense probe, based on this dot blot estimation, should be used in the hybridization reaction.

appropriate solution. All washes should be done under gentle shaking, making sure that sections are moving freely within the netwell.

1. Pour 1.0 Mg/mL proteinase K solution into the empty wells and incubate at 37°C until temperature stabilizes.

2. Transfer the netwells with sections into the proteinase K wells and incubate at 37°C for 30 min.

3. Transfer the sections to 4% paraformaldehyde and incubate 5 min at room temperature.

4. Transfer the sections to 2X SSC and incubate 5 min at room temperature.

5. While the sections incubate, add 2.5 ^L of acetic anhydride per milliliter of TEA-HCl buffer.

6. Transfer the sections to TEA-HCl/acetic anhydride and incubate for 10 min at room temperature.

7. Transfer the sections to 2X SSC and incubate at least 10 min at room temperature.

8. Place plates at 4°C until hybridization begins (preferably on the same day). 3.1.3.3. Hybridization

1. Prepare hybridization solution by diluting sense and antisense probes to an approximate concentration of labeled riboprobe of 500 ng/mL of hybridization buffer A (see Note 17). Approximately 500 ^L of solution per set of sections are needed.

2. Pipet 500 ^L of hybridization solution into each of the wells needed of a 24-well microplate.

3. Using an RNase-free glass rod or paintbrush, carefully transfer sections from 2X SSC to wells containing hybridization solution.

4. Cover the 24-well microplate with its lid and carefully seal with Parafilm. Make sure it is airtight.

5. Place the 24-well microplate inside a plastic container that has a DEPC-water-wet paper towel at the bottom, cover the plastic container with a hermetic lid, and incubate overnight on an orbital shaker in a high-humidity incubator at 60°C (see Note 19).

3.1.4. Post-Hybridization

1. Using an RNase-free glass rod or paintbrush, carefully transfer the sections from the hybridization mix in the 24-well microplate to the appropriate netwell in the 12-well microplate containing 50% formamide/50% 2X SSC at 60°C. Incubate 5 min at 60°C. When hybridizing with more than one probe, rinse the rod or the paintbrush with DEPC-treated water between probes.

2. Transfer the sections to a new well containing 50% formamide/50% 2X SSC and incubate 45 min at 60°C.

3. While this incubation takes place, warm up an appropriate volume of RNase buffer in a 37°C incubator. This should be done in the RNase area of the laboratory.

4. Transfer the sections to a new well containing 50% formamide/50% 2X SSC and incubate 15 min at 60°C.

5. Transfer the sections to 2X SSC and incubate 5 min at room temperature.

6. Transfer materials to the RNase area and add the appropriate amount of RNase A stock solution to the 37°C RNase buffer to a final concentration of 100 mg/L. Transfer the sections to the RNase solution and incubate 30 min at 37°C. Special care should be taken not to contaminate the RNase-free area of the lab after handling RNase A.

7. Transfer the sections to 50.0 % formamide/50% 2X SSC and incubate for 15 min at 60°C. Sections can be kept in 2X SSC at room temperature until temperature of incubator and formamide/2X SSC reach 60°C.

8. Transfer the sections to fresh 50.0% formamide/50% 2X SSC and incubate for 15 min at 60°C.

9. While incubation takes place, take advantage of the incubator temperature and prepare the blocking solution.

10. Transfer the sections to 0.4X SSC and incubate for 30 min at 60°C.

11. Transfer the section to buffer 1. Sections can be stored at 4°C overnight if necessary.

3.1.5. Immunohistochemical Detection of DIG

1. Wash sections in buffer 2 twice for 5 min each.

2. Transfer the sections to the blocking solution and incubate at room temperature for 1 h.

3. Transfer the sections to buffer 2 containing 1:500 peroxidase conjugated anti-DIG and incubate overnight at 4°C with gentle shaking. This step and the steps below are done in 24-well cell culture microplates.

4. Wash the sections twice for 5 min in buffer 2 and once for 5 min in buffer 1.

5. Incubate the sections for 8 min in 1:50 Cy3 TSA diluted in amplification buffer according to the manufacturer's instructions.

6. Wash the sections three times for 5 min in buffer 1.

7. Mount the sections on slides, let dry, rinse quickly with distilled water, let dry, and cover slip using a fluorescent dye anti-fade medium such as Prolong.

8. Cy3 labeling can be visualized under a fluorescence microscope with a Texas red filter or under a confocal microscope with the appropriate excitation wavelength (Fig. 3; see Note 20).

For double-labeling ISH combined with immunocytochemistry (Fig. 3B,C)

or double-labeling ISH, see Notes 21 and 22, respectively.

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