Rm 2155 rotary microtome

A male Dp16 mouse was crossed with a female WT C57BL/6J (The Jackson Laboratory). The female was checked daily for vaginal plugs. The female was separated from the male immediately in the morning (E0.5) of visual confirmation of vaginal plugs. The pregnant mice with E9.5 or E15.5 embryos were sacrificed by CO₂ inhalation and cervical dislocation. For JAKi treatment, pregnant mice received Tofacitinib (LC Laboratories) (10 mg/kg body weight) daily via intraperitoneal injection (i.p.) starting from E6.5 to the day before embryo harvest. The yolk sac (for E9.5) or a tail snip (for E15.5) was collected for genotyping. Embryos were then fixed in 2% paraformaldehyde (PFA) at 4°C overnight. Fixed embryos were stored in 70% ethanol before being embedded in paraffin. Embedded embryos were sectioned sagittally (E9.5) or transversely (E15.5) at 7μm thickness using a LEICA RM 2155 Rotary Microtome. Serial sections were collected throughout the developing heart to assess heart malformations. Representative sections were chosen for Hematoxylin and Eosin (H&E) staining as previously described.⁸¹ (link) Histological images were then captured using the Keyence BZ-X710 All-in-One Fluorescence Microscope.

On collection, embryos were fixed at 4 °C overnight in 2% paraformaldehyde (Ted Pella) diluted in 1× PBS and then stored in 70% ethanol before embedding in paraffin. Embedded embryos were sectioned transversely at 7 µm thickness using a LEICA RM 2155 Rotary Microtome. Serial sections were collected and then hematoxylin and eosin Y (H&E) staining was performed, followed by imaging with a Keyence BZ-X710 All-in-One Fluorescence Microscope. The investigators who sectioned embryos and performed H&E analysis were blind to embryo genotype. Number of animals per group were as follows: WT (n = 24, 12 male and 12 female), Dp16 (n = 18; 9 male, 7 female and 2 undocumented sex) and Dp16^2xIfnrs (n = 16, 4 male and 12 female).

Zebrafish juveniles and adults at 14, 30 and 90 dpf were euthanised with an overdose of MS-222 (300mg/L), fixed in 4% PFA and washed in PBST buffer. 30 and 90 dpf fish were decalcified in 0.5 M EDTA for one week with EDTA exchange every third day. Fish were transferred into 99.5% ethanol, followed by Xylene and embedded into paraffin. Sagittal head sections of 6μm were prepared with Leica RM2155 Rotary Microtome. Tissue sections were deparaffinised with xylene and re-hydrated through 99.5% to 70% ethanol series and transferred to water. Sections were then stained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA) for 30 sec followed by a brief water rinse and dehydration in 95% and 99.9% ethanol. Prior to mounting with VectaMount (Vector Laboratories, Burlingame, CA), slides were washed in Clear-Rite 3 (Richard-Allan Scientific, Kalamazoo, MI) three times for three minutes each. Sections were imaged with 40x objective on Hamamatsu NanoZoomer S60 Digital Slide Scanner.

Rat calvaria bone samples were subjected to decalcination in Osteofast 2 (Biognost, Zagreb, Croatia) for two days. After the decalcination process, the bone samples were embedded in paraffin blocks after which they were cut using a microtome (Leica RM 2155-Rotary Microtome, Leica instruments, Ballerup, Germany) to reach the centre of the defect. All specimens were cut through the sections of the parietal bone, with the crista temporalis and m. temporalis observed on the lateral margins of the convex surface of each section serving as orientation points. On the concave surface of some sections, the superior sagittal sinus was observed. Specimens were cut into 3–5 μm thick tissue sections and stained with hematoxylin and eosin (HE). Region of interest (ROI) is analogous to the margins of the bone defect and the zone within defect margins.