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Fast-track preparation of lung specimens for electron microscope observations of the pulmonary endothelial glycocalyx

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Abstract

The glycocalyx (GCX) covers the luminal surface of blood vessels and regulates vascular permeability. As GCX degradation predicts various types of vasculopathy, confirming the presence of this structure is useful for diagnosis. Since the GCX layer is very fragile, careful fixation is necessary to preserve its structure. We explored appropriate and feasible methodologies for visualizing the GCX layer using lung tissue specimens excised from anesthetized mice. Each specimen was degassed and immersed in Alcian blue (ALB) fixative solution, and then observed using electron microscopy. Specimens from septic mice were prepared as negative GCX controls. Using these immersion-fixed specimens, the GCX layer was successfully observed using both transmission and scanning electron microscopy; these observations were similar to those obtained using the conventional method of lanthanum perfusion fixation. Spherical aggregates of GCX were observed in the septic mouse specimens, and the GCX density was lower in the septic specimens than in the non-septic specimens. Of note, the presently reported methodology reduced the specimen preparation time from 6 to 2 days. We, therefore, concluded that our novel method could be applied to human lung specimens and could potentially contribute to the further elucidation of vasculopathies.

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Acknowledgements

We thank Mr. Oniki and Ms. Nagai for their support with the specimen preparation at Showa University. We thank KAC Co. for preparing the paraffin-embedded blocks and slides. We thank Ms. Myrna Harrod-Taniguti for the English proofreading.

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Authors and Affiliations

  1. Department of Perioperative Medicine, Division of Anesthesiology, Showa University School of Dentistry, Tokyo, Japan

    Mone Wakatsuki & Takehiko Iijima

  2. Center of Electron Microscopy, Showa University School, 1-5-8 Hatanodai, Shinagawa-Ku, Tokyo, 142-8555, Japan

    Takashi Takaki

  3. Department of Environmental Health, National Institute of Public Health, Saitama, Japan

    Akira Ushiyama

  4. Department of Anatomy, Showa University School of Medicine, Tokyo, Japan

    Takashi Takaki & Kazuho Honda

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  1. Mone Wakatsuki
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Correspondence to Takashi Takaki.

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Appendices

Appendix

Conventional perfusion fixation with lanthanum nitrate

SEM

A solution consisting of 2.5% glutaraldehyde (Nisshin-EM Co. Tokyo, Japan), 2% sucrose, and 2% lanthanum nitrate (Sigma-Aldrich Japan, Tokyo, Japan) was used as the perfusion fixative.

The mice were deeply anesthetized using an intramuscular injection of a cocktail of ketamine and xylazine. A perfusion pump was used to perform the injection at a steady rate of 1 mL/min for about 15–20 min. The lungs were then harvested and diced into 5 mm cubes.

Each cube was immersed in the perfusion solution for one day of fixation at 4 ℃. The specimens were then dehydrated using a graded ethanol series. Samples were freeze-cracked at the critical point, osmium-coated, and observed using SEM at an acceleration voltage of 15 kV, a spot size of 40, and the high vacuum mode [8, 27,28,29,30].

TEM

Perfusion-fixed lungs were diced into 1-mm cubes and immersed in 2.5% glutaraldehyde, 2% sucrose, and 2% lanthanum nitrate fixing solution at 4 ℃ for one day. The specimens were then washed with 30 mM HEPES (DOJINDO LABORATORIES, Japan) and fixed in 2% osmium tetroxide at 4 ℃ for 2 h.

Specimens were dehydrated in a graded ethanol series and embedded in epoxy resin. After ultra-thin sectioning, the specimens were stained with 2% uranyl acetate and 2.7% lead citrate and were observed using TEM at an acceleration voltage of 80 kV [2, 9, 17].

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Wakatsuki, M., Takaki, T., Ushiyama, A. et al. Fast-track preparation of lung specimens for electron microscope observations of the pulmonary endothelial glycocalyx. Med Mol Morphol 56, 239–249 (2023). https://doi.org/10.1007/s00795-023-00360-1

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