Killik

Biopsies were collected in 0.9% NaCl saline solution with penicillin (100 UI/mL) immediately after surgery, macro-sectioned, embedded in Killik (BioOptica, Milan, Italy), and snap-frozen in 2-methyl butane/liquid N₂. Since all specimens were surgical waste, to avoid damage or alterations biasing the results, only IMA with surrounding perivascular adipose tissue were included in the study, whilst the fragments from artery skeletonized during grafting procedure were discarded.
Before being snap-frozen, a subset of sample rings from IMA (n = 3) and CPL (n = 6) was kept in culture, as previously described [38 (link)], and 2 rings/fragment were either untreated or submitted to treatment with A740003, added to culture medium at 100 μM for 24 h (t1) or 72 h (t3).
Numbered serial cryosections (10 μm thick) were obtained and dedicated either to confocal videomicroscopy experiments (pair numbers) or to histology and immunofluorescence (odd numbers). For blood flow videomicroscopy experiments, the sections were collected on glass coverslips coated with bovine fibronectin (50 μg/mL in phosphate buffered solution, R&D Systems Inc., Minneapolis, MN, USA).
Reagents, whereas not differently indicated, were purchased from EuroClone S.p.A., Milan Italy and Sigma-Aldrich Saint Louis, MO, USA.

The right eyes of OXYS rats from control and SkQ1-treated groups (n = 4) were removed and fixed in fresh 4% paraformaldehyde in PBS for 2 h, washed three times in PBS, and then cryoprotected in graded sucrose solutions (10%, 20%, and 30%). Posterior eyecups were embedded in Killik (Bio-Optica), frozen, and stored at −80 °C. Tissue slices (10 µm thick) were prepared on a Microm HM-505N cryostat at −20 °C, transferred onto Polysine^® glass slides (Menzel-Glaser), and stored at −20°C. After several washes in PBS with 0.1% Triton X-100 (PBST), the slices were incubated for 1 h in 5% BSA in PBST, followed by overnight incubation at 4 °C with the rabbit antibodies to Aβ_1–42 (1:50 dilution, Abcam) and to p-S6 (1:150 dilution, Cell Signaling Technology). After three washes in PBST, the tissue slices were incubated with the secondary antibody conjugated with Alexa Fluor^® 488 (Abcam) at a dilution of 1:300 for 1 h and next washed with PBST. The slices were coverslipped with the Fluoroshield mounting medium supplemented with DAPI (Abcam) and examined under an Axioplan 2 microscope (Zeiss). For acquisition of each image, all imaging parameters were the same.

A set of animals (P15 n = 15, P90 n = 14) was employed for the histological analysis of the sensorimotor cortex in order to better characterise the SCI model. Either 12 h or 3 days after injury both P-15 and P-90 mice were anaesthetised with 3% isoflurane vaporised in O₂/N₂O 50:50 and transcardially perfused with 0.1 M PBS, pH 7.4 followed by 4% PFA in PBS. The brain and spinal cord (C6 level) were dissected and post-fixed for 2 h at 4°C in the same fixative solution. Samples were transferred overnight into 30% 0.1M PBS at 4°C then embedded in cryostat medium (Killik, Bio-Optica, Milan, Italy), frozen at −70°C in 2-methylbutane and cut on the cryostat (Microm HM 550) in coronal and transverse 50 μm thick sections for brain and spinal cord, respectively. Sections were collected into 1× PBS prior to immunofluorescence reactions and Fluoro-Jade C (FJC; Histo-Chem Inc., Jefferson, AR, United States) staining.

After the abovementioned washing procedure, the stigmas were embedded with a mounting medium (Killik, Bio-Optica, Milano, Italy) and then sliced with a Leica CM 1510 S cryostat (Leica microsystems^®, Wetzlar, Germany) to obtain 150-µm-thick cross-sections. These (n = 28 ± 3) were transferred over polysine glass slides (Menzel Gläser™, Thermo Fisher Scientific, Waltham, MA, USA), dehydrated at room temperature, and then washed with dH₂O to remove the mounting medium. The slices were then stained with aniline blue for five minutes, washed to remove the excess of stain, and observed with the abovementioned epi-fluorescence microscopy system. Pollen germination over the stigmatic surface was evaluated on the digital images were acquired with the Fiji software. For each sample, 193 ± 32 pollen grains were counted from randomly chosen images to calculate the percentage of germinated pollen (approx. 1100 pollen grains counted per treatment).

Gonadal tissues of specimens from S and SM were histologically examined to determine the stage of gonadal development. After embedding in Killik (Bio-Optica, Milano, Italy) medium, sections of 8–10 μm were obtained using Leica cryostat (Wetzlar, Germany). The histology prepared slides were stained with haematoxylin and eosin method and inspected using the optical light microscope (Olympus Co., Tokyo, Japan).

To evaluate probiotic presence in the gut, bacteria were labeled with wheat germ agglutinin-Alexa Fluor 555 conjugate (WGA-AF555) (Thermo Fisher) for 10 min, followed by extensive washes. A dose of 10⁹ CFU bacteria was administered to Lewis rats, and gut samples were excised after 30–60 min, washed with PBS, fixed with paraformaldehyde (PFA, 4% in PBS) for 24 h and then transferred in sucrose (30% in PBS) for cryopreservation. Samples were included in Killik (Bio-Optica) and kept at −80°C, pending analysis. Serial 10 μm thick cryosections were stained with Hematoxilin and Eosin (images digitalized with ScanScope, Aperio technologies) or with the following antibodies: mouse anti-vimentin mAb (V9, Dako), mouse anti-cytokeratin mAb (MNF116, Dako), mouse anti-CD11c mAb (8A2; ThermoFisher), mouse anti-CD3 mAb (G4.18, eBioscience), followed by species-specific Alexa Fluor 488-conjugated secondary antibodies. Isotype control stainings were routinely performed in the immunofluorescence procedures. Nuclei were stained with DAPI (Thermo Fisher Scientific). Single plan and z-scan images were captured via confocal microscopy and Structured Illumination microscopy (SIM), using a 100X APO-TIRF (NA 1.49) objective, with 3D optical sectioning. Images were processed with Fiji software (20 (link)).

For the histological analysis of the spinal cord, at P12 animals (N = 3 WT PBS; 17 SMA PBS; 6 WT D-JNKI1; 29 SMA D-JNKI1) were anesthetized by gaseous anesthesia and perfused transcardially with phosphate buffer (0.1 M PB, pH 7.4), followed by cold 4% paraformaldehyde (PFA) in 0.1 M PB (pH 7.4). The spinal cord was removed from the vertebral column at the lumbar level (L1–L4) and postfixed in 4% PFA for 2 h. The tissue was then cryoprotected in 30% sucrose solution in 0.1 M PB buffer overnight, then embedded, and frozen in cryostat medium (Killik, Bio-Optica, Milan, Italy). The spinal cord was cut into transverse, 40 μm thick, free-floating sections that were stored in an antifreeze solution (30% ethylene glycol, 30% glycerol, 10% PB; 189 mM NaH₂PO₄; 192.5 mM NaOH; pH 7.4) and stored at −20°C until being used.
For the histological examination of quadriceps muscles, another cohort of pups (five animals per group) were sacrificed by cervical dislocation. Fresh quadriceps muscles were rapidly collected, embedded in cryostat medium and cut on the cryostat. Coronal and parasagittal muscle slices (20 μm thick) were cut and collected directly onto 4% gelatin-coated glasses.