Skeletal muscle samples from mouse hind legs were collected bilaterally at birth or 6 dpi, fixed with 4% paraformaldehyde, and embedded in paraffin after dehydration. Paraffin-embedded tissue sections of 3 to 5 μm were prepared and stained with hematoxylin and eosin (H&E) or used for immunohistochemistry. H&E images were obtained by using optical microscopy at a magnification of 10× (Olympus BX40), and images were acquired using Leica Application Suite 3.8 software. For scoring, mock-infected or ZIKV-infected skeletal muscle tissues stained with H&E from at least five different animals were classified for the presence of necrosis, cellular infiltrate, and fiber atrophy by a researcher blind to the experimental condition, according to the following criteria: 0, absence; 1, some indicative; 2, moderate; 3, intense; and 4, very intense.
For immunohistochemistry of ZIKV dsRNA, sections of skeletal muscle were stained as previously described (8 (link)) with primary mouse anti-dsRNA antibodies (SCICONS J2) at a 1:200 dilution. Stained tissue sections were analyzed under identical conditions to allow comparison between immunoreactivity ODs. Slides were imaged on a Sight DS-5M-L1 digital camera (Nikon, Melville, NY) connected to an Eclipse 50i light microscope (Nikon).
For immunohistochemistry of ZIKV dsRNA, sections of skeletal muscle were stained as previously described (8 (link)) with primary mouse anti-dsRNA antibodies (SCICONS J2) at a 1:200 dilution. Stained tissue sections were analyzed under identical conditions to allow comparison between immunoreactivity ODs. Slides were imaged on a Sight DS-5M-L1 digital camera (Nikon, Melville, NY) connected to an Eclipse 50i light microscope (Nikon).
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