Prolong antifade mountant with dapi

For sterile muscle injury, mice were anesthetized with isoflurane and 50 μL cardiotoxin (12 × 10^–6 M, 217503, Millipore) injected in the TA muscle. Muscles 8 days after injury were snap frozen in nitrogen-chilled isopentane. 8 μm cryosections were cut and stained with H&E. For each analysis, more than 10 slides (per condition/group) containing 6 muscle sections/sample were used, and myofibers in the injured area were counted and measured with a Mirax digital scanner and HALO software (68 (link)). For adult mouse studies, muscles were fixed in 10% formalin and embedded in paraffin for sectioning. Laminin staining (RB-082, Thermo Fisher Scientific) was used to outline fibers, and picrosirius red was used for collagen accumulation. Image analysis was performed using ImarisX64 software (Bitplane AG), and fiber sizing was calculated using minimal Feret’s diameter (69 (link)). For after weaning juvenile mouse studies, cryosections from soleus muscles were mounted in ProLong antifade mountant with DAPI (Thermo Fisher Scientific). Fiber type, size, and central nucleation were quantified with analysis via SMASH (70 (link)) and ImageJ (NIH). Antibodies used were mouse anti-MHC2A (1:50, SC-71, Developmental Studies Hybridoma Bank) and mouse anti-eMHC (1:10, BF-45, Developmental Studies Hybridoma Bank).

Spleens were dissected, embedded in OCT compound (Tissue-Tek), and snap-frozen for cryosectioning (6 µm). Slides were then incubated in 100% ethanol to fix for 5–10 min (4 °C), followed by rehydration in PBS for 5 min (4 °C). The sections were blocked with 10% normal goat serum for 20 min at room temperature (RT), and then rinsed with PBS. The tissues were incubated with primary antibodies for 1 h at RT and then washed 3× in PBS (2 min for each wash). When secondary antibodies were used, the tissue was further incubated with the secondary antibody for 1 h at RT, again followed by three washes in PBS (2 min for each wash). The slides were finally mounted in Prolong Antifade Mountant with DAPI (Thermo Fisher), imaged on a Leica SPE confocal microscope using LAS X software, and analyzed using Fiji (ImageJ).

Immunofluorescence assays were performed using blood smears fixed with 4% paraformaldehyde for 30 min followed by washing in PBS and permeabilisation in 0.1% Triton-X100 for 10 min. Slides of HA-tagged parasite lines were blocked overnight at 4°C in 3% bovine serum albumin/PBS and then labelled with rabbit anti-HA high affinity (1:250) and Alexa Fluor 488-conjugated α-rabbit IgG (1:5,000) (Thermo Fisher Scientific). The smears were mounted in ProLong Antifade mountant with DAPI (Thermo Fisher Scientific). For live cell imaging parasites were stained with Hoechst 33342 (New England Biolabs), transferred to poly-L-lysine-coated μ-slides VI (Ibidi, Martinsried, Germany). Both live and fixed preparations were viewed with a Nikon Ti E inverted microscope using a 100x oil immersion objective and imaged with an ORCA Flash 4.0 CMOS camera (Hamamatsu). Images were acquired and processed using the Nikon Elements Advanced Research software package.