Leitz 1512 rotary microtome

Cross-sectional samples of each meniscus were taken for histological analyses (Fig. 1). To verify the decellularization process, cell-seeded menisci were dissected and fixed in 10 % formalin for 24 h, followed by two washes in phosphate-buffered saline (PBS), ethanol dehydration, and paraffin embedding. Menisci were sectioned into 10-μm thick slices with a Leitz 1512 rotary microtome (Leica, Wetzlar, Germany), and sections were stained with 4,6-diamidino-2-phenylindole (DAPI) to identify residual nuclear debris. Additional sections were stained with fast green/safranin O with a hematoxylin counterstain to visualize collagen organization, proteoglycans, and cellular infiltration in both the “red-red zone” and “white-white zone” of the menisci.Fig. 1

Diagram of meniscus sample preparation. Five-millimeter plugs were taken from the anterior and posterior for material property testing. Biochemical samples were taken beneath the surface of the meniscus. Histological samples were taken in cross sections and imaged in the “red-red zone” towards the exterior of the meniscus and at the “white-white zone” towards the interior of the meniscus

Formalin fixed-paraffin embedded placental tissue was sectioned at 5 μm through the mid-line using a Leitz 1512 rotary microtome (Leica). Placental sections were stained with haematoxylin and eosin to assess gross morphological structure and layer proportions. Placental glycogen was stained using a periodic acid Schiff (PAS) stain. Briefly, sections were dewaxed and rehydrated before incubation with 0.5% periodic acid (Sigma, UK) for 90 min. Sections were then washed and incubated in Schiff's reagent (Sigma, UK) for 15 min, before dehydrating and mounting in DPX. The percentage of positive magenta staining was measured by a blinded operator using Image J software.
Total placental glycogen content was determined using the protocols of Lo et al. (1970) and Rampon et al. (2008) with some modifications [24 ,25 (link)]. Briefly, 10 mg of placental tissue was homogenised in 30% KOH saturated with Na₂SO₄ (Sigma, UK) and incubated at 100 °C for 30 min, followed by cooling on ice. Glycogen was precipitated in 100% ethanol and separated by centrifugation at 1,000×g for 30 min. Glycogen standards were prepared using Rabbit Liver Glycogen (G8876; Sigma, UK), with concentrations ranging from 400 μg/ml to 12.5 μg/ml. Samples and standards were combined with 5% phenol with H₂SO₄ (Sigma, UK) to initiate a colorimetric change. Absorbance was read at 490 nm on Benchmark Bio-Rad microplate reader.