Dfc7000 t camera

Paraffin-embedded tissue sections were deparaffinized in xylene and then rehydrated. Antigen- retrieval was performed by heating with citrate-based antigen-unmasking solution (Vector). For experiments using HRP detection, endogenous peroxidase was quenched using 3% hydrogen peroxide in methanol. Nonspecific antigen binding was blocked using donkey serum. Sections were incubated with primary antibodies at the dilutions shown below before application of secondary antibodies. Samples containing EdU were also counter stained per protocol using the Click-IT^™ EdU Cell Proliferation Kit (Invitrogen #C10339). For HRP detection, Vectastain ABC and DAB kits were used. Sections were counterstained using hematoxylin, dehydrated and cover slipped. Samples using immunofluorescent antibodies were counter-stained with DAPI before analysis.
For PRoX1 nuclei counting, non-serial murine lung tissue sections approximately 350 μm apart were stained as described above. Nuclei were visualized via light microscopy using a Nikon Eclipse E200 microscope or Leica DM4 B microscope with a Leica DFC7000 T camera. Immunofluorescent samples were imaged using a Leica DM4 B microscope with a Leica DFC7000 T camera. We would also like to acknowledge S10oD030269 instrumentation for supporting the microscopic analysis. See Table 1 for list of antibodies and dilutions.

Histopathological evaluation was conducted on three mice at each time‐point: 3rd, 5th and 14th day post‐injury. Small fragments from the left gastrocnemius muscles were collected and fixed by immersion in 4% glutaraldehyde, post‐fixed in buffered 1% OsO₄ with 1.5% K₄Fe(CN)₆ (potassium ferrocyanide–reduced osmium), dehydrated in graded ethanol series and further processed for epoxy resin embedding (AGAR 100). One‐micrometre‐thick sections (semi‐thin sections) were stained with 1% toluidine blue and examined by light microscopy for morphological analysis with Leica DM 600. Images were recorded using a Leica DFC7000 T camera.

Immunohistochemistry (IHC) staining was performed as previously described for frozen sections^{45 (link)}. Primary and secondary (HRP or florescent labeled) antibodies were acquired commercially (Supplementary Table 1). Images were acquired on a Leica DM4 B LED microscope using a DFC7000 T camera, and LAS X Software. Fluorescent confocal images were acquired on a Leica multi-photon confocal microscope at 630× with 3× digital zoom. Digital image analysis was performed using ImageJ (v1.52k; National Institutes of Health, Bethesda, MD). Length, area, and integrated density of microglia and albumin were calculated by drawing regions of interest in different fields per biological replicate. DAB quantifications and integrated density calculations were performed as before^{45 (link)}.