Vs120 fluorescent slide scanner

Formalin-fixed muscles were bisected and paraffin-embedded, and 10-μm cross sections were taken at the midbelly region. Sections were deparaffinized, rehydrated and subjected to Masson’s trichrome stain. In regard to immunohistochemitry, deparaffinized and rehydrated10-μm cross sections were subjected to antigen retrieval by heating in Rodent Decloaker solution (BioCare Medical, Pacheco, CA) at 80 °C for 30 min. Sections were blocked in 5% goat serum in PBS and incubated in rabbit anti-laminin (Sigma) diluted in 1:100 in blocking solution. Sections were then incubated in goat anti-rabbit secondary antibody conjugated to Alexa 488 (Life Technologies) diluted 1:200 in blocking solution. For detection of type II fibers sections were further incubated in anti-fast (type II) MHC conjugated to alkaline phosphatase (Sigma, St. Louis, MO) diluted 1:50 in PBS for 60 min at room temperature. Whole stained sections were scanned on an Olympus VS120 fluorescent slide scanner.

RNAscope in situ hybridisation was performed on brain cryosections using an RNAscope Fluorescent Multiplex Assay V2 (Advanced Cell Diagnostics) to simultaneously visualise up to 3 mRNA targets using mouse-specific probes directed against genes listed in Supplementary Table 8. A 3-plex positive control probe targeting the housekeeping genes Polr2a, PPIB, and UBC (cat. no. 320881; Advanced Cell diagnostics) was used to verify the mRNA quality and assay procedures. A 3-plex probe targeting the bacterial gene dapB (cat. no. 320871), which is not expressed in eukaryotes, was used as a negative control. Slides with cryosections were treated according to the RNAscope Fluorescent Multiplex Assay user manual using FITC, Cy3, and Cy5 as fluorophores. After in situ hybridisation, the slides were counterstained with DAPI, cover-slipped, and scanned under a 20× objective in an VS-120 fluorescent slide scanner (Olympus).