*2.10. Fluorescence in Situ Hybridization*

FISH experiments were performed on cryostat sections using sterile solutions and materials. Diethylpyrocarbonate (DEPC) was added to phosphate-buffered saline (PBS) and water 1 mL/L to inactivate RNase enzymes; solutions were shaken vigorously and autoclaved.

Sections were dried for 2 h at room temperature (RT), well washed in 1× DEPC/PBS and treated with 10 μg/μL Proteinase K (Sigma-Aldrich, St. Louis, MO, USA) 1:200 in DEPC/PBS for 10 min. Proteinase K action was then inactivated by two washes in 2 mg/mL glycine, 5 min each. Sections were post fixed in 4% PFA for 20 min and well washed in 1× DEPC/PBS at RT. Thereafter, the prehybridization was carried out in a hybridization solution (HB) containing 50% formamide, 25% 20× SSC, 50 μg/mL Heparin, 10 μg/mL yeast RNA, 0.1% Tween 20, and 0.92% citric acid at 55 ◦C (riboprobes) and 42 ◦C (LNA probes) for 1 h. All probes were denatured for 10 min at 80 ◦C and sections were then incubated, in HB containing riboprobes concentration of 500 pg/μL, ON at 55 ◦C and LNA probes concentration of 2 ng/μg, ON at 42 ◦C. Post-hybridization washes were carried out at 55 ◦C as follows: 2 × 20 min in 1× SSC, 2 × 10 min in 0.5× SSC, and then in 1× DEPC/PBS at RT. Sections were blocked in blocking solution (BS) containing 10% normal sheep serum heat inactivated and 0.5% blocking reagent (Roche, Hamburg, Germany) for 1 h at RT. After, sections were incubated in a 1:2000 dilution of anti-digoxigenin Fab fragments conjugated with alkaline phosphatase (Roche) in BS, 2 h at RT. Sections were well washed in 1× DEPC/PBS. The chromogenic reaction was carried out by using Fast Red tablets (Sigma-Aldrich) in Tris buffer and incubating the slides at RT in the dark and were observed every 20 min until the signal detection (1–10 h depending on the probe used). After the signal was developed, sections were washed in 1× DEPC/PBS at RT and mounted with Fluoreshield Mounting Medium with DAPI as counterstaining for the nuclei.
