A cryostat

A cryostat (Leica) was used to produce 10 μm-thick sections of fresh-frozen tissue. After fixation in − 30 °C methanol for 10 min, hematoxylin–eosin was used to stain the sections. Immunohistochemistry was then performed on the sections by treatment with 10 μg/ml of anti-CD31 goat polyclonal antibody (R&D systems, AF3628) for 16 h at 4 °C. Whole amount CD31 staining of E8.5 embryos were performed using the same antibody for 24 h at 4 °C. After washing, staining was done using Goat IgG VisUCyte HRP Polymer Detection Reagent (R&D systems) according to the manufacturer’s protocol. Most anatomical analyses and evaluation were done using embryos (E8.5) or 6 weeks old littermate pairs (wild type vs. TG/+) (n = 5). Retinas of 11-week-old littermates pairs (n = 2) were stained with 2 μg/ml of Alexa-Flour 488 conjugated Isolectin GS-IB4 (Thermo Fisher) for 1 h at 4 °C for bifurcation angle analysis.

Mice were anesthetized and perfused with cold PBS. The whole brain was fixed with 4% paraformaldehyde solution for 24 h at 4 °C. Next, the fixed brain was incubated in 30% sucrose solution for 72 h at 4 °C. Brain tissue was cut to a 30-μm thickness using a cryostat (Leica). Brain sections were washed in PBS and then incubated with 70% formic acid solution for antigen retrieval. The sections were immersed in a solution with BSA, 0.3% Triton X-100, and 5% horse serum overnight at room temperature to block nonspecific binding and enhance antibody permeability. Next, the sections were incubated with primary antibodies against the target proteins overnight at 4 °C. The primary antibodies included anti-Iba-1 (Wako; 1:1000), anti-GFAP (Invitrogen, 1:1000), anti-SOX2 (Abcam, 1:100), anti-Calbindin (Cell Signaling, 1:200), anti-4G8 (Biolegend, 1:500), anti-DCX (Santa Cruz, 1:200), and anti-synaptoporin (SYSY, 1:250). After reaction with primary antibodies, the sections were incubated with secondary antibodies (Invitrogen; 1:400) for 1 h at room temperature. Finally, the sections were incubated with DAPI (0.4 μg/ml) to stain nuclei. Mounted brain sections were imaged by an LSM700 confocal laser scanning microscope (Carl Zeiss), and images were analyzed by ImageJ software.

Mice were deeply anesthetized with sodium pentobarbital (70 mg/kg, i.p.) and subjected to brief trans-cardiac perfusion with PBS and subsequently 4% paraformaldehyde (PFA, in 0.1 M phosphate buffer, pH 7.4). Brains were isolated and incubated in 4% PFA at 4°C overnight for post-fixation. Fixed brains were then incubated in 30% sucrose (in PBS) at 4°C for at least 36 h for cryoprotection. Tissues were embedded and frozen on Tissue-Tek optimum cutting temperature (O.C.T.) compound with dry ice. Twenty or 35 μm-thick coronally-cut brain sections were prepared using a cryostat (Leica) and kept in PBS complemented with 0.1% sodium azide.

Fresh mouse testes were embedded in CMC compound (FINETEC), sectioned at 4 μm using a cryostat (Leica), and then incubated in PBST that contained 1 μg/ml proteinase K at 37°C for 5 min. After post-fixation with 4% PFA, the sections were rinsed in PBST three times and then hybridized overnight with 1 μg/ml DIG-labeled cRNA probe dissolved in hybridization buffer (50% formamide, 5x saline-sodium citrate, 1% SDS, 50 μg/ml heparin, and 50 μg/ml yeast RNA) at 65°C in a moist chamber. The sections were then washed in 50% formamide, 5x SSC, pH4.5, and 1% SDS at 65°C for 30 min, and in TBST three times at room temperature for 5 min each. After blocking by 0.5% blocking reagent (Roche) in TBST at room temperature for 30 min, the sections were incubated in sheep anti-DIG antibody conjugated to alkaline phosphatase in the blocking buffer at 4°C overnight. The colorization was subsequently developed by BM purple AP substrate solution (Roche Applied Science) according to the manufacturer’s instructions.

Transgenic embryos were collected at different stages. The date of reimplantation of the one cell stage embryos in pseudo-pregnant females was considered as embryonic day 0.5 (E0.5). Embryos were dissected and a tissue block containing the stomach; pancreas and duodenum was removed and fixed by immersion in 4% PFA for 24 h. Tissues were cryoprotected by incubation for 48 h in 15% sucrose prepared in PBS and next embedded in 15% sucrose, 7% gelatin prepared in PBS then frozen in isopentane at -50°C. 10 μm sections were performed using a cryostat (Leica). Whole embryos were fixed in 4% PFA for 20 min and stained for lacZ reporter activity as described and embedded in BSA-gelatin [49 (link)]. 300-μm sections were cut with a Vibratome (Leica VT1000S) as described [50 (link)].

Matrigel plugs were fixed for 1–2 hours with PFA 4 % in PBS and then dehydration was performed by sucrose gradient method at 4 °C with sucrose 10 % in PBS for 1 hour, 15 % for 1 hour and 30 % overnight. Pieces were then frozen in liquid nitrogen. Muscle samples were frozen by submersion in isopentane cooled with liquid nitrogen. Cryosections were obtained after embedding samples in OCT (optimum cutting temperature) compound (Kaltek, Padua, Italy) and using a cryostat (Leica) to produce sections. For specific antibody concentrations and conditions, see Table 1. Phase-contrast and bright-field pictures were taken by using an Olympus IX71 inverted microscope (Olympus, Tokyo, Japan). Immunofluorescence pictures were acquired by using a Leica DMI 6000B inverted microscope.

A digoxigenin-labelled riboprobe was synthesized from linearized plasmid pCR4-TOPO (Invitrogen; nucleotides 2705–3558 of NM_001163847). Tissue samples were snap frozen in Optimal Cutting Temperature and 15 μm sections were cut using a cryostat (Leica) and mounted onto Superfrost Plus slides (Avantor). Probe hybridization, washing and signal detection using an alkaline phosphatase-conjugated anti-DIG antibody was carried out as previously described (20 (link)).