Bx51trf microscope

Cryosections of vastus lateralis muscle from the immobilized limb after 14 days of post-immobilization recovery were stained for collagen expression using picrosirius red (PSR) staining, as described previously [28 (link)]. Fifteen-micrometer sections of muscle were fixed in ice-cold acetone, washed in PBS, and stained in Bouin’s reagent (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The sections were then rinsed in water, stained with PSR (Sigma Direct Red 80, Sigma-Aldrich) for 1 h, and rinsed in acidic water (5 mL glacial acetic acid in 1 L of water) and a picric alcohol (10% picric acid, 20% ethanol) rinse. The slides were then cleared in xylene and mounted with Permount medium (Sigma-Aldrich). Slides were imaged in a blinded manner using an Olympus DP72 Digital Camera System mounted to an Olympus BX51 TRF Microscope (Olympus, Center Valley, PA, USA), and the collagen fiber area was determined using custom image analysis software (programmed by RGB; full code can be accessed at https://github.com/rbudnar/PSR).

Translocations of the NUTM1 gene at 15q14 were analyzed via FISH using the ZytoLight SPEC NUTM1 Dual Color Break Apart Probe. Samples were first fixed for 24 h with 10% neutrally buffered formalin at room temperature, embedded at a temperature below 65°C, and sliced into 2–4 μm thick sections via microtome. Sample pretreatment was conducted based on the instructions provided with the ZytoLight FISH-Tissue Implementation Kit, and an Olympus BX51TRF microscope (Olympus, Japan) was used for FISH signal analysis with a triple-pass filter (DAPI/Green/Orange; Vysis). Samples were deemed to have a positive FISH signal when there was a distance between red and green signals of ≥2 signal diameters.

We used cell lines from the College of American Pathologists (CAP), FFPE samples from clinical STS patients, and synthetic fusion gene fragments as the reference sample set for accuracy validation (Table S2). 17 well-characterized cell lines with known gene fusions from the CAP SARComa or RNA sequencing proficiency test (PT) were also included. Besides, 48 fusion constructs were synthesized by GENEWIZ Inc. (Suzhou, China), with the junction sequence of fusions as a template. 14 FFPE sections from STS patients were included in this part, on which fluorescence in situ hybridization (FISH) assays had been performed using break-apart probes to detect one of the chromosomal rearrangements: EWSR1, FOXO1, FUS, or SS18 translocation. FISH signals were assessed under an Olympus BX51TRF microscope (Olympus, Japan). Signals were considered to be split when the distance between red and green signals ≥ 3 signal diameters. Cells without the rearrangement had two sets of red and green fusion signals indicating intact chromosomes. For each case, a minimum of 200 tumor nuclei were evaluated by two independent operators. A positive result was reported when ≥ 10% of the tumor nuclei had break-apart signals.
The reference material was analyzed using RNA‐NGS, then assessed if they aligned with the expected results.

Floral buds of 01-20 and HE-DGMS01-20 cabbage at different developmental stages were collected, fixed in FAA solution (50% ethanol, 5% acetic acid, 3.7% formaldehyde), and stored at 4 °C overnight. The fixed floral buds were dehydrated through an ethanol solution series of 70%, 80%, 90% and 100% followed by clearing in a xylene solution series of 25%, 50%, 75% and 100% and then embedded in paraffin (Paraplast High Melt, Leica). The samples were cut into 8–10 μm sections using a microtome (RM2255, Leica, Wetzlar, Germany) and then mounted on poly-L-lysine-coated glass slides (P0425-72AE; Sigma‒Aldrich). A 200-bp cDNA fragment from Ms-cd1 was subsequently synthesized and inserted into a pGEM-T Easy vector (Promega, Madison, WI, USA). Digoxigenin (DIG)-labeled sense and antisense probes were produced from T7 and SP6 polymerase using a DIG Northern Starter Kit (12039672910, Roche) following the manufacturer’s instructions. RNA ISH with the sense and antisense probes was performed, observed under a BX-51TRF microscope (Olympus, Tokyo, Japan) and imaged with a microcolor charge-coupled device camera (UCMOS05100KPA, ToupTek Photonics, Hangzhou, China).