Proximal jejunum and distal ileum sections were used for histologic examination. They were fixed with 4% paraformaldehyde–phosphate-buffered saline overnight, then dehydrated, and embedded in paraffin blocks. After that, a section of 5 μm was cut and mounted on slides. The sections were further deparaffinized and hydrated and then stained with hematoxylin-eosin (H&E) for microscopy. Microphotographs were taken with a DM300 microscope (Leica, Germany). Villus length and crypt depth were performed using Image J software. A minimum of 20 well-orientated villi and associated crypts from at least seven different fields per animal were measured.
Dm300 microscope
Manufactured by Leica
Sourced in Germany
The Leica DM300 is a compact and versatile microscope designed for routine laboratory applications. It features high-quality optics, including a 10x eyepiece and objectives ranging from 4x to 100x magnification, providing a wide range of magnification options. The DM300 is equipped with a professional-grade illumination system and mechanical stage for precise specimen positioning. Its durable construction and easy-to-use controls make it a reliable and efficient tool for a variety of laboratory tasks.
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8 protocols using dm300 microscope
1
Histological Analysis of Intestinal Morphology
2
Histological Analysis of Liver Tissue
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Liver tissues were fixed in 4% paraformaldehyde-PBS overnight, then were dehydrated and embedded in paraffin blocks. After that, a section of 5 μm was cut and mounted on slides. The sections were further deparaffinized, hydrated, and then stained with hematoxylin and eosin (H&E) for histological analysis. Images were captured using a DM300 microscope (Leica, Weztlar, Germany).
3
Retinal Vasculature Trypsin Digestion
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The retinal vasculature was prepared through trypsin digestion, as previously described [17 (link)]. Briefly, retinas were fixed overnight using 4% paraformaldehyde, followed by incubation in 3% trypsin 250 (BD Biosciences, San Jose, CA, USA) on a gentle shaker at 37 °C. After each 30-min trypsin incubation interval, the retinas were rinsed with PBS until the internal limiting membrane was digested, leaving only the retinal vasculature. Subsequently, the retinal vasculature was meticulously mounted onto a glass slide and stained using periodic acid-Schiff’s base (PAS)-hematoxylin (Sigma-Aldrich, MO, USA). A Leica DM300 microscope was employed to capture retinal images, with acellular capillaries quantified across 10 randomly selected fields by two independent blinded investigators.
4
Imaging Nanoparticle Uptake in RAW 264.7 Cells
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RAW 264.7 cells were cultured on slides in 6-cm dishes for 12 h, followed by co-culture with nanoparticles (5 mg/mL) for 6 h. The cells were incubated with stains (DAPI for nuclei staining, DIO for nanoparticles staining, and WGA for membrane staining) at 4 °C for 1 h in the dark. The stains included DAPI (DA002, Leagene Biotechnology, Shanghai, China), DIO (D4007, US Everbright®Inc., Suzhou, China), and WGA (MP6326-1MG, MKBio, Shanghai, China). A Leica DM300 microscope was used to capture the images. The Leica Application Suite X software 3.0.0.15697 was used for quantitative analysis.
5
Immunohistochemical Analysis of Tumor Proliferation and Apoptosis
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After the mice were sacrificed, the tumor tissues were dissected and fixed in formaldehyde. Following paraffin embedding, sectioning (5 µm), deparaffinization, incubation in 3% H2O2 for 10 min at room temperature, antigen retrieval by boiling in 0.01 M sodium citrate buffer (pH 6.0) for 15 min, the tissues were blocked with 10% FBS in PBS for 30 min at room temperature and stained with an anti-ki67 antibody (1:200) at 4°C overnight. Subsequently, the sections were incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (1:200) for 30 min at room temperature, incubated with DAB at for 1-2 min room temperature, counterstained with hematoxylin, dehydrated and stabilized with mounting medium (cat. no. ab64230; Abcam). For TUNEL assay, an in situ Cell Death Detection kit (Roche Molecular Diagnostics) was used to detect apoptosis according to the manufacturer′s instructions following deparaffinization. The sections were counterstained with hematoxylin for 3 min at room temperature following dehydration and stabilizing with mounting medium. The slides were visualized under a Leica DM300 microscope (magnification, ×200; Leica Microsystems GmbH), and 5-10 fields of view per sample were quantified using Leica Application Suite X software 3.0.0.15697 (Leica Microsystems GmbH).
6
Histochemical Analysis of PEDV in Piglets
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We fixed 1 cm × 1 cm × 1 cm jejunum of each piglet in 4% paraformaldehyde (Beijing Biotopped Technology Co., LTD, Beijing, China) to obtain the paraffin sections. According to the reported procedure mentioned in the reference (11 (link)), each fixed jejunum was cut and stained with Hematoxylin-eosinstaining (HE). Immunohistochemistry (IHC) was involved in dewaxing paraffin-embedded tissue slices and heating them in a pressure cooker. Antigen retrieval was performed on the slices using a citrate antigen retrieval solution (Beyotime Biotech. Inc., Shanghai, China) in a microwave oven. Endogenous peroxidase activity was blocked using 3% H2O2. Sections were incubated with normal goat serum buffer for 20 min and then with a mouse anti-PEDV N mAb (diluted 1:200) for 1 h at room temperature. Sections were then incubated with a secondary antibody before a DAB Horseradish Peroxidase Color Development Kit (JK GREEN Tech INC., Beijing, China) was used to develop the color reaction. The cell nucleus was re-stained with Mayer’s hematoxylin for 1 min. The brown staining of PEDV on intestinal villous epithelial cells was classified as positive staining. Pathological changes in the intestinal tissue samples were observed under a Leica DM300 microscope (Leica Microsystems, Wetzlar, Germany). In addition, we used ImageJ software for statistical analysis of the proportion of positive signal area.
7
Quantifying Intestinal Morphology
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Samples of duodenum, jejunum and ileum fixed with 4% paraformaldehyde were dehydrated with 70, 80, and 90% alcohol, and cleaned by treating with xylene solution. Then, the tissues were embedded with paraffin and sectioned at a thickness of 3 to 5 μm. After staining with Periodic Acid Schiff, images were captured using a DM300 microscope (Leica Microsystems, Wetzlar, Germany). The villus height (VH ) and crypt depth (CD ) (Luo et al., 2021 (link)) were observed under an optical microscope, and VH to CD ratio (VCR ) were calculated. Ten points of each section were randomly selected for measurement and the average value was taken.
8
Intestinal Morphology Analysis
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Paraformaldehyde-fixed intestine samples were dehydrated, and then embedded in paraffin. The 5 μm consecutive sections of each sample were prepared for morphological observations. After the sections were stained with hematoxylin eosin (H&E), images were captured using a DM300 microscope (Leica Microsystems, Wetzlar, Germany). Villus length (VH) and crypt depth (CD) were measured, and then the ratio of VH to CD (VH/CD) was calculated out [26 (link)].
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