Transverse serial cross sections (7 μm) were obtained using a cryostat maintained at −20°C. Sections were then blocked (1× PBS/5% normal serum/0.3% Triton X-100), and laminin primary antibody (Thermo Fisher Scientific, MA1-06100, 1:100) was diluted in dilution buffer (1× PBS/1% BSA/0.3% Triton X-100) and incubated overnight at 4°C. Three washes with PBS were performed before applying secondary antibody (Abcam, ab175476, 1:1,000) at room temperature for 1 h, and ProLong diamond antifade mountant was used (Thermo Fisher Scientific, 36961). Stained slides were digitalized with the Zeiss Apotome.2 microscope with a ×20 objective (Hamamatsu), and myofiber CSA was semiautomatically quantified using SMASH application on MATLAB software (v. R2021b) (17 (link)). In addition, we investigated the lipid content through oil red O staining. Only lipids outside muscle fibers were quantified (MATLAB software v. R2021b) to exclude myocellular triglyceride content and then reflect IMAT deposits. Finally, collagen area was investigated through modified Masson’s trichrome staining (18 ) and quantified with MATLAB software (v. R2021b).
Apotome 2 microscope
Manufactured by Hamamatsu Photonics
The Apotome.2 microscope is a structured illumination microscope (SIM) system developed by Hamamatsu Photonics. The core function of the Apotome.2 is to provide optical sectioning and improved resolution in fluorescence microscopy, enabling the visualization of fine structures within a sample.
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3 protocols using apotome 2 microscope
1
Histological Analysis of Skeletal Muscle
2
Quantification of Muscle Apoptosis
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Transverse serial cross sections (7 μm) were obtained using a cryostat maintained at −20°C. Then, an in situ cell death detection kit (Merck, 11684795910) based on the TUNEL method was used to quantify single‐cell apoptosis according to the manufacturer's instructions. At least five muscle sections per patient were analysed from different parts of the muscle specimen. For each section, all TUNEL‐positive signals were manually quantified and DAPI was used to ensure that the TUNEL‐positive signals were co‐localized with a nucleus. Results are expressed as an absolute value of TUNEL‐positive nuclei per muscle section.
In addition, the total myofibre CSA as well as the type I and type IIa myofibre CSA were assessed. We performed these analyses according to the protocol of Murach et al.
15 All slides were digitalized with a Zeiss Apotome.2 microscope with a ×20 objective (Hamamatsu).
In addition, the total myofibre CSA as well as the type I and type IIa myofibre CSA were assessed. We performed these analyses according to the protocol of Murach et al.
15 All slides were digitalized with a Zeiss Apotome.2 microscope with a ×20 objective (Hamamatsu).
3
Visualizing Nematode Dauer Recovery
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A sample of 1% SDS-treated nematodes was placed on 2% agar pads and anaesthetized in a drop of 0.2% levamisole, and a cover slip placed on top. Images were captured using a Zeiss ApoTome.2 microscope with a Hamamatsu digital camera C13440 ORCA-Flash4.0 V3 and Zen software. Recovering dauers were identified by virtue of one or more of the following characteristics: increased diameter of the pharynx, twitching in the terminal bulb of the pharynx (recovery of pharyngeal pumping function) and increased body length.
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