Rats under deep anesthesia were perfused with 0.1 M phosphate buffer (PBS) and 4% paraformaldehyde (PFA) afterwards via the ascending aorta. The L4-6 spinal cord was collected and postfixed in the same fixative overnight at 4°C then replaced with 20, 30% sucrose in 0.1 M PBS, respectively, overnight. In a cryostat, transverse free-floating spinal cord pieces (25 μm) were sliced (Leica, CM1950). The sections were treated for 5 min with antigen retrieval solution (C1035, Solarbio, China), then blocked for 1 h at room temperature with 5% albumin bovine serum in 0.3% Triton X-100, followed by overnight at 4°C with the primary antibody. Afterward, the slices were treated for 2 h at room temperature with the matching secondary antibody. For double immunofluorescence staining, spinal segments were incubated with a combination of two primary antibodies (from two different species) and then processed with a mixture of two second antibodies. The staining of the slices was imaged by ECLIPSE Si (Nikon, Japan). The detailed information on the antibodies used above is listed in Table 2 .
Eclipse si
Manufactured by Nikon
Sourced in Japan
The ECLIPSE Si is a microscope system designed for laboratory use. It features optical components and a sturdy frame to support various microscopic investigations.
Automatically generated - may contain errors
Lab products found in correlation
Cm1950, by Leica (2 mentions)
C1035, by Solarbio (1 mentions)
Fitc conjugated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Cy3 conjugated goat anti mouse secondary antibody, by Jackson ImmunoResearch (1 mentions)
Digital sight 1000, by Nikon (1 mentions)
Cm3050, by Leica (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
3 protocols using eclipse si
1
Immunofluorescence Staining of Rat Spinal Cord
2
Immunohistochemistry of Spinal Cord and ACC
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Following defined survival durations, the rats were terminally anesthetized and perfused with normal saline via the ascending aorta, then immersed in 4% paraformaldehyde in 0.1 M phosphate buffer. Following perfusion, the spinal cord and ACC were removed and postfixed for 12 h in the same fixative, subsequently replaced with 20, 30, and 40% sucrose phosphate buffered saline throughout one night. Transverse spinal cord (20 m) and ACC (20 m) slices were produced for immunofluorescence staining using a cryostat (Leica, CM1950). After washing with PBS, the sections were blocked for 1 h at 37°C with 5% goat serum in 0.3 percent Triton X-100 and incubated overnight at 4°C with the primary antibody. The sections were treated for 2 h at 37°C with a combination of goat anti-mouse Cy3-conjugated secondary antibody (1:200, Jackson ImmunoResearch, Amish, PA) and goat anti-rabbit FITC-conjugated secondary antibody (1:400, Jackson ImmunoResearch). ECLIPSE Si was used to visualize the staining of the slices (Nikon, Japan). The primary antibodies listed in Table 2 were utilized.
3
Microscopic Analysis of Duodenum Tissue Tension
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Microscopic observations were performed to investigate the fibrous structure of the duodenum when the tissue is under tension. The trouser-shaped duodenum tissues were pulled apart and fixed in 10% formalin (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) using pins on a silicone mat (Fig. 2a). The tissues were fixed for 16 h at room temperature. Tissues were then embedded in embedding medium (O.C.T. Compound, Sakura Finetek Japan, Tokyo, Japan) and cryosectioned at 10 µm thickness using a cryostat (CM3050S, Leica Biosystems, Wetzlar, Germany) (Fig. 2b). The sections were placed on microscopic slides (APS-03 15, Matsunami Glass Ind., Ltd., Osaka, Japan) for observation. Sections were stained with hematoxylin and eosin (ScyTek Laboratories, Utah, USA) following the protocol suggested by the supplier. The stained sections were observed using an optical microscope (Eclipse Si, Nikon Corporation, Yokohama, Kanagawa, Japan) and photographs of the microscopic images were taken using a C-mount camera mounted onto the microscope (Digital Sight 1000, Nikon Corporation, Yokohama, Kanagawa, Japan).
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