Mouse tissues were fixed in 10% buffered formalin (Fisher Scientific, catalog SF100-20) overnight and embedded in paraffin. Sections (8 μm thick) were stained with H&E. Images were acquired on a TissueFAXS Whole Slide Scanning System (TissueGnostics) using a 20x objective. Nuclei quantification was performed with Image J.
For immunofluorescence staining, muscle sections were de-paraffinized in xylene and rehydrated using a graded ethanol series culminating with PBS. Following antigen retrieval using a programmable pressure cooker with “target retrieval solution”, pH 6.0 (Dako), tissue sections were blocked with 10% goat serum in PBS. The slides were then stained for CD8 (1:500, D4W2Z, Cell Signaling Technology), granzyme B (1:40, AF1865, R&D Systems), F4/80 (1:100, MCA497R, Bio-Rad), and OVA (1:500, AB1225, Millipore Sigma) for 16 hours at 4°C. Species-specific secondary antibodies conjugated to Cy5 or Cy7 fluorophores were used and incubated for one hour at room temperature in the dark. Sections were washed, counterstained with DAPI (100 ng/ml) and mounted using FluorSave (Calbiochem) mounting medium. Images were acquired on a Leica SP8 laser scanning confocal microscope using a 40x oil-immersion objective. Quantification of the fluorescent signals of the respective markers was performed using QuPath (69 (link)).
For immunofluorescence staining, muscle sections were de-paraffinized in xylene and rehydrated using a graded ethanol series culminating with PBS. Following antigen retrieval using a programmable pressure cooker with “target retrieval solution”, pH 6.0 (Dako), tissue sections were blocked with 10% goat serum in PBS. The slides were then stained for CD8 (1:500, D4W2Z, Cell Signaling Technology), granzyme B (1:40, AF1865, R&D Systems), F4/80 (1:100, MCA497R, Bio-Rad), and OVA (1:500, AB1225, Millipore Sigma) for 16 hours at 4°C. Species-specific secondary antibodies conjugated to Cy5 or Cy7 fluorophores were used and incubated for one hour at room temperature in the dark. Sections were washed, counterstained with DAPI (100 ng/ml) and mounted using FluorSave (Calbiochem) mounting medium. Images were acquired on a Leica SP8 laser scanning confocal microscope using a 40x oil-immersion objective. Quantification of the fluorescent signals of the respective markers was performed using QuPath (69 (link)).
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