Two-micrometer-thick sections of the paraffin-embedded terminal ileum were sliced, dewaxed, rehydrated and incubated with 3% H2O2 using standard methods. Sections were incubated with a 1:1000 dilution of anti-zonula occludens-1 (ZO-1) antibodies overnight at 4°C and then incubated with a 1:100 dilution of a FITC-conjugated goat anti-rabbit secondary antibody (Beyotime, Shanghai, China) for 1 h at room temperature. Terminal ileum sections were finally scanned using a Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
Lsm t pmt confocal microscope
Manufactured by Zeiss
Sourced in Germany
The LSM T-PMT confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a scanning laser and a sensitive photomultiplier tube (PMT) detector, enabling high-resolution, three-dimensional imaging of a variety of samples.
Automatically generated - may contain errors
Lab products found in correlation
Cy3 conjugated secondary antibody, by Beyotime (3 mentions)
Nanozoomer digital pathology system, by Hamamatsu Photonics (2 mentions)
Fitc conjugated goat anti rabbit secondary antibody, by Beyotime (1 mentions)
Ab pas, by Solarbio (1 mentions)
Occludin, by Proteintech (1 mentions)
Zonula occludens 1 zo 1, by Proteintech (1 mentions)
Hrp linked secondary antibody, by Agilent Technologies (1 mentions)
Tnf α, by Wuhan Servicebio Technology (1 mentions)
C11 bodipy, by Thermo Fisher Scientific (1 mentions)
Cellrox green, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
15 protocols using lsm t pmt confocal microscope
1
Immunofluorescence Analysis of Tight Junctions
2
Histopathological Evaluation of Intestinal Inflammation
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Upon euthanasia, distal colon segments were immediately postfixed in 10% formalin. The tissue sample was embedded in paraffin and stained with hematoxylin and eosin. The degree of enteritis, which uses a scoring system as previously described (Chen et al., 2008 (link)), consists of epithelial cell damage (score: 0–3), congestion/edema (score: 0–3), and neutrophil infiltration (score: 0–3). Two independent pathology professors double-blindly evaluated these sections. Alcian blue and periodic acid-Schiff (AB-PAS) staining were performed with the instructions (Solarbio). The sections were stained with p-mTOR, beclin1, zonula occludens-1 (ZO-1), occludin, and claudin-1 antibodies as previously described (Chung et al., 2014 (link)). Images were managed using the Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
3
Histological Analysis of Distal Colon
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Distal colon segments in the same defined region were collected carefully and were transferred immediately to 10% neutral-buffered formalin. After paraffin embedding, colon fragments were generated into 5 μm cross-sections. The sections were stained with hematoxylin and eosin (H&E) and then scanned by a NanoZoomer Digital Pathology system (Hamamatsu Photonics, KK, Japan) for further histopathological analysis. At least three slides were randomly selected and observed by a blinded pathologist. The histological activity index (HAI) is an established scoring system based on inflammatory cell infiltration (1–3), both in terms of severity and extent, and intestinal architecture (1–3), which includes epithelial changes and mucosal architecture, as previously described (Erben et al., 2014 (link)).
The colon sections were stained with the intestinal barrier markers ZO-1 and Occludin (both Proteintech, Rosemont, IL, United States) with a previously described standard immunofluorescence analysis process (Chung et al., 2014 (link)). Images were captured and visualized using a Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
The colon sections were stained with the intestinal barrier markers ZO-1 and Occludin (both Proteintech, Rosemont, IL, United States) with a previously described standard immunofluorescence analysis process (Chung et al., 2014 (link)). Images were captured and visualized using a Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
4
Immunohistochemical Evaluation of Liver and Colon
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Paraffin-embedded liver sections were stained for F4/80 (anti-active macrophage) (Abcam, Cambridge, United Kingdom) and α-SMA (fibrosis hallmark) with immunohistochemistry (IHC) staining procedures as previously detailed[14 (link)]. Briefly, liver sections were incubated with a specific primary antibody, followed by incubation with horseradish peroxidase (HRP)-linked secondary antibody (Dako, Glostrup, Denmark) and 3,3’-diaminobenzidine; the sections were then scanned with the NanoZoomer Digital Pathology system. Image-Pro Plus software was used to count F4/80+ cells and quantitatively analyze the staining intensity of α-SMA as previously described[13 (link)]. Six fields of view were randomly selected in each section.
Likewise, paraffin-embedded colon sections were stained for Zonula occludens-1 (ZO-1) (intestinal barrier hallmark) (Proteintech, Rosemont, IL, United States) with standard immunofluorescence staining procedures as previously detailed[15 (link)]. Briefly, sections were incubated with the rabbit polyclonal ZO-1 antibody, followed by incubation with Texas Red-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA, United States) and 4’,6-diamino-2-phenyl indole (DAPI), and images were captured using a Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
Likewise, paraffin-embedded colon sections were stained for Zonula occludens-1 (ZO-1) (intestinal barrier hallmark) (Proteintech, Rosemont, IL, United States) with standard immunofluorescence staining procedures as previously detailed[15 (link)]. Briefly, sections were incubated with the rabbit polyclonal ZO-1 antibody, followed by incubation with Texas Red-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA, United States) and 4’,6-diamino-2-phenyl indole (DAPI), and images were captured using a Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
5
Confocal Microscopy of RANKL-Induced NF-κB Activation
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All cells were pre‐treated with Sch for 24 hours and then stimulated by RANKL 50 ng/mL for 30 minutes, followed by a fixation with paraformaldehyde for 10 minutes and blocked with 5% goat serum for 1 hour. Then, cells were incubated with anti‐p65 antibody (CST, 1:200) overnight at 4°C and with a Cy3‐conjugated secondary antibody (Beyotime, 1:500) for 1 hour the next day. Images were acquired with a Zeiss LSM T‐PMT confocal microscope (Zeiss).
6
Immunofluorescence Staining of Cells
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According to protocols, pretreated cells were fixed with methanal and blocked with serum. Then, the cells were incubated with specific primary antibodies overnight and with secondary antibodies for 1 h. Images were acquired with an LSM T-PMT confocal microscope (Zeiss).
7
Immunohistochemical Analysis of Colon Tissues
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Immunohistochemistry and immunofluorescence were performed in the colon tissues as previously reported (Lv et al., 2014a (link)). Sections were stained with primary antibodies against (TNF-α, MCP-1, NF-KB p65, phospho-p65, and ZO-1) (Servicebio, Wuhan, China). Images were analyzed with the NanoZoomer Digital Pathology system (Hamamatsu Photonics K.K., Japan). A total of 10 vision fields were randomly selected from each slide and the mean positive area was calculated and analyzed by Image J software. The disruption and disorganization of ZO-1 immunostaining were visualized with a Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
8
Quantifying Lipid Peroxidation with C11-BODIPY
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The lipid peroxidation was detected by C11-BODIPY (Invitrogen). Briefly, cells were harvested after different treatments and incubated with 2 uM C11-BODIPY for 20 min at 37°C. Images were acquired with an LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
9
Mitochondrial and Cytoplasmic ROS Detection
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MitoSOX red (Invitrogen, CA, USA) and CellROX green (Invitrogen) were used to detect ROS in mitochondria and cytoplasm, respectively. Briefly, BMMs were cultured with 5 µM MitoSOX and CellROX dyes for 30 mins at 37°C. Then, images were obtained by an LSM T‐PMT confocal microscope (Zeiss).
10
Phospho-STAT3 Immunofluorescence Assay
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All cells were treated with 5 mM NaB for 24 h and 25 ng/ml IL-6 for 30 min, fixed for 15 min, and blocked with 5% goat serum for 1 h. Then, the cells were incubated with anti-p-STAT3 antibody (CST, 1:200) overnight at 4 °C and with a Cy3-conjugated secondary antibody (Beyotime, 1:500) for 1 h the next day. Images were acquired with a Zeiss LSM T-PMT confocal microscope (Zeiss, Jena, Germany).
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