Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

NOTE: Ethics Statement: Procedures involving animal subjects were approved by the animal welfare committee of the University of Hamburg and the University of Saarland.

1. Preparation and Fixation of Embryonic Tissue

1. Arrange all the equipment needed to dissect out the embryos and prepare solutions for subsequent fixation of the tissue before sacrificing the animals.
   NOTE: Always prepare a fresh 4% paraformaldehyde (PFA) solution (4% PFA in 0.1 M phosphate buffered saline (PBS), adjust pH to 7.4).
2. Euthanize timed pregnant female mouse with an ethically approved procedure.
3. Transfer the mouse onto a platform. Wet the ventral side of the abdomen with 70% ethanol and make a vertical incision using sharp scissors to expose the abdominal cavity.
4. Identify the embryonic chain and pull it out carefully using blunt forceps and place it into ice-cold PBS.
5. Remove the individual embryos from the embryonic chain using fine scissors and forceps. Remove extra tissue around the embryos and wash them in ice-cold PBS.
6. Take a small tail biopsy (before transferring the embryo into PFA) and incubate it in tail lysis buffer for the gender identification and tracer allele genotyping PCR.
7. Start from the embryo at the most lateral side and transfer each embryo separately into a tube containing ice-cold 4% PFA on ice on a shaker.
8. Soak the embryos at the age of E13.5 or younger for 1.5 hr on ice with constant shaking. Soak the embryos at age E14.5 and E15.5 for 2.5 hr on ice with constant shaking.
9. For embryos age E16.5 or older, decapitate and remove the outer skin before soaking the head in PFA solution. Heads of E16.5 embryos can be soaked for 4.5 hr on ice with constant shaking.
10. After appropriate fixation, wash the embryos three times with ice-cold PBS. Transfer the embryos into 30% sucrose in PBS solution at 4 °C until they sink to the bottom.

2. Freezing

1. Arrange all the materials required before starting the freezing process: cryo-mold, cryo-gloves, featherweight entomology forceps, marker pen and optimal cutting temperature compound (O.C.T.)
2. Label the cryo-mold with a permanent alcohol-resistant marker (mention the orientation of tissue, date and genotype). Prepare a slush of crushed dry ice and 100% ethanol in an ice bucket. Place a glass beaker inside containing isopentane. Wait for 10 min for the isopentane to cool down.
3. Meanwhile remove the tissue from the sucrose solution, wipe off excess sucrose around the tissue using laboratory wipes. Transfer the tissue into a pre-labeled cryo-mold with sufficient O.C.T. to cover the tissue. Avoid any air bubbles in the O.C.T.; especially around the tissue.
4. Orient the tissue in O.C.T. using featherweight entomology forceps to avoid tissue damage. Start freezing by transferring the cryo-mold into the pre-cooled isopentane bath. Do not splash isopentane into the O.C.T. as it will not freeze properly. Wrap the samples in aluminum foil and store at -80 °C.

3. Cryosectioning

1. Cut 14 µm thin serial sections in series’ of five using a cryostat and collect on SuperFrost® Plus glass slides for immunofluorescence (IF) analysis. Store the slides at -80 °C until utilized.

4. Tracer visualization Using the Tyramide Signal Amplification (TSA) Protocol

1. Prepare the buffers and reagents required for staining. Always freshly prepare the Tris-NaCl-Tween (TNT) buffer on the day of use.
   1. Prepare TNT buffer using 100 ml of 1 M Tris-HCl pH 7.5, 30 ml of 5 M NaCl and ddH₂O up to a final volume of 1 L. Add 500 µl of Tween 20 using a 1 ml pipette. Add the Tween 20 slowly and flush the pipette up and down several times to ensure that all Tween 20 is in solution.
   2. Prepare Tris-NaCl-Blocking (TNB) buffer using 100 ml of 1 M Tris-HCl pH 7.5, 30 ml of 5 M NaCl and ddH₂O up to a final volume of 1 L. Add 5 g Blocking Reagent (provided with the kit) slowly in small increments to the buffer while stirring. Heat the solution gradually to 55 °C with continuous stirring to completely dissolve the Blocking Reagent (this should not take longer than 30-60 min). To achieve homogenous heating use a water bath at 55 °C. The solution will appear milky. Bring the solution to room temperature before using. Aliquot and store at -20 °C for long-term storage.
   3. Reconstitute biotin tyramide reagent (provided with the kit) in Molecular Biology/HPLC-grade DMSO. Depending upon which kit is used the appropriate amount of DMSO may vary. Store the stock solution at 4 °C.
      NOTE: Sections from animals heterozygous for the respective Cre and R26 alleles can be used for circuit analysis. Use sections from littermates carrying one Cre or one R26 allele, respectively, as negative controls. Treat negative control slides exactly like slides from double heterozygous animals. In addition, include one control slide from a double heterozygous animal but leave out the TSA reagent (unamplified control).
2. Using a sharp fresh scalpel remove excess O.C.T. surrounding the tissue sections and mark the boundary around the sections with a PAP pen.
3. Dry the slides for 2-3 min at room temperature (RT). Wash the slides at RT 3x with TNT for 5 min each. Incubate the slides at RT for 30 min in 0.3% H₂O₂ solution in ice-cold methanol to quench endogenous peroxidase activity.
4. Wash 3x with PBS for 5 min each at RT. Incubate for 10 min in 0.5% Triton-X 100 in PBS for tissue permeabilization. Wash the slides at RT 3x with TNT for 5 min each.
5. Block with TNB (blocking solution, 200-300 µl per slide) for 30 min at RT in a humidified chamber. Make sure all the sections are completely covered by TNB. Do not let the slides dry out in between the steps.
6. Drain off excess blocking buffer and apply the primary antiserum recognizing the tracer (1:1,000 goat anti-WGA for BL, 1:15,000 rabbit anti-GFP for GTT) diluted in TNB for 2 hr at RT or overnight at 4 °C. Make sure to use enough volume to completely cover the tissue section (200-300 µl per slide).
   NOTE: The primary antisera dilutions recommended here may have to be adjusted.
7. Wash the slides at RT 3x with TNT for 5 min each with agitation.
8. Incubate the sections with biotinylated secondary antiserum recognizing the host of the first antiserum (1:500 horse anti-goat IgG for anti-WGA, 1:5,000 goat anti-rabbit IgG for anti-GFP in TNB for 1 hr at RT).
9. Wash 3x with TNT at RT for 5 min each with agitation. Incubate in 1:100 streptavidin (SA)-conjugated horseradish peroxidase (SA-HRP, provided with the kit) in TNB for 30 min at RT in a humidified chamber. Wash the slides at RT 3x with TNT for 5 min each. Meanwhile thaw the TSA.
10. Dilute TSA 1:50 in TSA amplification diluent (provided with the kit; use the TSA kit for the visualization of BL and the TSA+ kit for GTT). Incubate the tissue sections in diluted TSA for precisely 10 min at RT. Dilute TSA just before use, ideally during the previous washing step, do not use any remaining diluted TSA reagent for future experiments; always prepare fresh.
11. Wash the slides at RT 3x with TNT for 5 min each with agitation. Incubate with 1:500 Alexa Fluor® 546 streptavidin conjugate in TNB for 30 min at RT. Wash the slides at RT 3x with TNT for 5 min each with agitation.
12. Incubate for 10 min in 5% bisbenzimide solution in PBS at RT for nuclear staining. Wash the slides at RT 3x with TNT for 5 min each with agitation. Mount with Fluromount G and perform epifluorescence or confocal microscopy.
    Troubleshooting: Efficacy of tracer transfer will depend on the expression level of the ROSA26 locus in the producing cells (determined by the Cre driver line used) and on the neural circuit analyzed (e.g. number of neurons producing the tracer, convergence etc.). Therefore the primary antisera dilutions recommended here may have to be adjusted to prevent low or excess signal. Background should always be analyzed using appropriate control slides (see above).

5. τlacZ Staining for Embryonic Sections

1. Prepare the buffers and reagents required for staining. Always freshly prepare the LacZ buffer C before use.
   1. Prepare 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) stock solution (40 mg/ml) by adding 40 g of X-gal to 1 ml 70% dimethylformamide (DMF). Cover with aluminium foil and store at -20 °C for long-term storage.
   2. Prepare nitro blue tetrazolium (NBT) stock solution (50 mg/ml) by adding 50 g of NBT to 1 ml 70% DMF. Cover with aluminium foil and store at -20 °C for long-term storage.
   3. Prepare LacZ fixative using 80 µl of 25% glutaraldehyde, 5 ml of 4% PFA, 20 µl of 1 M MgCl₂, 100 µl of 500 mM EGTA, 1 ml of 1 M phosphate buffer pH 7.4 and ddH₂O up to a final volume of 10 ml.
   4. Prepare LacZ buffer A using 1 ml of 1 M MgCl₂, 5 ml of 500 mM EGTA, 50 ml of 1 M phosphate buffer pH 7.4 and ddH₂O up to a final volume of 500 ml.
   5. Prepare LacZ buffer B using 1 ml of 1 M MgCl₂, 5 ml of 500 mM EGTA, 5 ml of 1% sodium deoxycholate, 100 ml of 10% NP-40, 50 ml of 1 M phosphate buffer pH 7.4 and ddH₂O up to a final volume of 500 ml.
   6. Prepare LacZ buffer C by adding 10 µl of NBT stock solution and 12.5 µl of X-gal stock solution to 1 ml of LacZ buffer B. Make fresh just before use and cover with aluminium foil.
2. Dry the slides at RT at least for 10-15 min (meanwhile prepare the slides with a PAP pen).
3. Fix the section with LacZ fixative for 2 min at room temperature in a humidified chamber inside a chemical fume-hood. Wash 3 times with PBS supplemented with 2 mM MgCl₂.
4. Rinse the slides with LacZ buffer A. Wash the slides with LacZ buffer A 3 times for 10 min each at RT. Wash 2 times with LacZ buffer B for 5 min each. Incubate the slides in LacZ buffer C at 30-37 °C for 6 hr or overnight in a humidified chamber.
   NOTE: Add sufficient volume of LacZ buffer C; slides should not dry out during incubation.
5. After appropriate incubation wash the slides with PBS supplemented with 2 mM MgCl₂. Rinse sections briefly with ddH₂O. Coverslip with Mayer’s glycine gelatin. Suitable for a light microscope equipped with a differential interference contrast (DIC) imaging set-up.

6. Gender and Tracer Genotyping

1. Prepare tail lysis buffer using 1 ml of 1 M Tris-HCl pH 8.5, 100 µl of 500 mM EGTA, 200 µl of 10% SDS, 400 µl of 5 M NaCl and ddH₂O to make up a final volume of 10 ml.
2. Add 500 µl of tail lysis buffer to each Eppendorf tube containing a tail biopsy. Add proteinase K to a final concentration of 100 mg/ml. Incubate overnight on a shaker at 55 °C.
3. Centrifuge samples at 17,000 x g for 10 min at RT. Transfer the supernatant to a new centrifuge tube. Add 500 µl of isopropanol to the supernatant. Mix for at least 5 min by inverting the tubes up and down.
4. Spin in centrifuge at 17,000 x g for 5 min at RT. Pour off the supernatant. Add 200 µl of 70% ethanol. Shake for at least 5 min.
5. Centrifuge at 17,000 x g for 5 min. Remove supernatant with a Pipetman (do not pour off). Dry the pellet in a warm chamber (37 °C) for 15-30 min.
6. Resuspend pellet in 100 µl of low TE pH 8.0 or water using wide-bore filtered tips. Put in 55 °C for 1 hr and at 37 °C overnight for proper dissolving. Store at 4 °C.
7. Use 1 µl of DNA for PCR reaction (50 μl reaction mixture containing 1 μl of tail DNA, 2.5 μl DMSO, 10 pM of each primer, 25 μM of each dNTP, and 1M betaine). PCR primers for genotyping and gender identification are listed in the Materials table.