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A1r a1

Manufactured by Nikon
Sourced in Japan

The Nikon A1R/A1 is a high-performance confocal laser scanning microscope designed for advanced biological research. It provides high-resolution imaging capabilities for a wide range of applications. The system features a modular design, allowing for flexible configuration to meet the specific needs of the user's research.

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48 protocols using a1r a1

1

Identification of Sequestered Nematocyst Types

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The type of sequestered NCs was identified using two methods: (1) analysis of the thin and ultrathin sections of the cnidosac, and (2) analysis of NCs in discharged cnidosacs using the Transmission Detector Analyzer option in the confocal laser scanning microscope Nikon A1R-A1 (Nikon Corporation, Tokyo, Japan). For this purpose, we followed the classification and NC descriptions provided by Östman [21 , 56 ]. In both identification methods, the three characters were used: (1) the shape of the NC; (2) the presence, size, and shape of the shaft, and (3) the spine pattern of the shaft and the tubule (which is clearly visible in both TEM and CLSM). Due to the restrictions of this methodology, we did not identify types of NCs (e.g., p-/b-mastigophores, homotrichous/heterotrichous euryteles, etc.) as that would require SEM studies of discharged NCs. The precise number of specimens studied is shown in Table S1 (columns TEM + CLSM).
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2

Histological Assessment of Liver Damage

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Following reperfusion, about 0.25 g liver samples were fixed with 10% buffered formalin for 24 h at room temperature (pH=7.2; cat. no. G2161; Beijing Solarbio Science & Technology, Co., Ltd., Beijing, China), embedded in paraffin and cut into 5-µm sections for histological analysis via hematoxylin-eosin staining (H&E; hematoxylin staining for 5–15 min and eosin staining for 1–3 min; all performed at room temperature). Sections were analyzed under a confocal microscope (magnification, ×200; Nikon A1R/A1; Nikon Corporation, Tokyo, Japan) and images were obtained. A total of 6 fields of view per section were randomly selected for the assessment of liver damage. Numerical assessment of liver damage was conducted according to the histological criteria for assessment of liver damage (32 (link)).
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3

TUNEL Assay for Apoptosis Detection

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Cells cultured in a Millicell® EZ SLIDE 8-well glass (Merck Millipore, Darmstadt, Germany) were washed with PBS three times, fixed with 4% paraformaldehyde for 30 min, washed with PBS again and treated with permeabilization solution (1% Triton X-100™ (Sigma-Aldrich; Merck Millipore) in PBS) for 5 min. Subsequently, incubated with terminal deoxynucleotidyl transferase-containing reaction mixture, which was part of the One Step TUNEL Apoptosis Assay kit (Beyotime Institute of Biotechnology, Shanghai, China), for 60 min at 37°C in the dark. Cells were washed with PBS three times and stained with streptavidin-tetramethylrhodamine for 30 min at 37°C in the dark. Subsequently, cells were washed with PBS three times and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min in the dark. Finally, the samples were visualized using a confocal laser scanning microscope (Nikon A1R/A1; Nikon Corporation, Tokyo, Japan).
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4

Immunofluorescence and Staining of hMSCs

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The hMSCs cultured on the substrates were fixed using 4% paraformaldehyde for 15 min and then permeabilized with 0.05% Triton X‐100 (Sigma‐Aldrich) for 10 min at room temperature. After blocking the slides with 2% fetal bovine serum (FBS, Sigma‐Aldrich) and 2% bovine serum albumin (BSA, Sigma‐Aldrich) in phosphate‐buffered saline (PBS, Sigma‐Aldrich) for 1 h, primary antibodies (Table S1, Supporting Information) were added and incubated at 4 °C overnight. Alexa Fluor‐488 conjugated anti‐mouse IgG secondary antibody (Invitrogen, 1:200) was used as the secondary antibody. The fluorescent dye rhodamine‐phalloidin (PHDR1 Cytoskeleton, 1:200) and 4′,6‐diamidino‐2‐phenylindole (DAPI, H‐1200 VECTOR,1:1000) were used to observe the cytoskeleton and nuclei, respectively. Confocal laser scanning microscopy (CLSM, Nikon A1R/A1) was used to observe the corresponding protein locations.
For alkaline phosphatase (ALP) staining, hMSCs were fixed in 4% paraformaldehyde for 20 min and stained using the BCIP/NBT alkaline phosphatase color development kit (Beyotime) for 30 min at room temperature, avoiding natural light. For Alizarin Red S staining, hMSCs were fixed in 4% paraformaldehyde for 20 min and then incubated in 3‐mm ARS (Sigma‐Aldrich) for 30 min at room temperature.
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5

Liver Histology Examination Protocol

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Following reperfusion, about 0.25 g liver samples were fixed with 10% buffered formalin for 24 h at room temperature (pH=7.2; cat. no. G2161; Beijing Solarbio Science & Technology, Co., Ltd.), then put into 75, 80, 85, 90, 95 and 100% alcohol for 1 h respectively, so as to fully dehydrate the tissues. Following dehydration, the tissues were soaked in 50% alcohol + 50% xylene solution for 30 min, and then soaked in 100% xylene until they became transparent. The transparent samples were soaked in liquid paraffin at 60°C twice (1 h each) until the paraffin had completely infiltrated. The samples after paraffin immersion were embedded in paraffin wax and cut into 4-µm sections for histological analysis via hematoxylin-eosin staining (H&E; hematoxylin staining for 5–15 min and eosin staining for 1–3 min; all performed at room temperature). Sections were analyzed under a confocal microscope (magnification, ×200; Nikon A1R/A1; Nikon Corporation) and images were obtained. A total of 6 fields of view per section were randomly selected for the assessment of liver damage. Numerical assessment of liver damage was conducted according to the histological criteria for assessment of liver damage.
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6

Detecting Cardiomyocyte Apoptosis and Oxidative Stress

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After 4 hours' reperfusion, the heart was removed. Formalin-fixed heart tissues were then embedded in paraffin and approximately 5 μm thick sections were cut. TUNEL staining was used to detect cardiomyocyte apoptosis. The section images were taken by a confocal microscopy (NIKON A1R/A1, Nikon, Japan). For dihydroethidium (DHE) staining, fresh frozen sections were incubated with 10 μmol/L of DHE (Beyotime Biotechnology, Shanghai) at 37°C for 30 minutes, and then DAPI was applied. Images were acquired by fluorescence microscope.
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7

Immunofluorescence and ROS Detection Assay

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Cells were washed with PBS 1X and fixed with PFA 4%. Immunofluorescence studies were performed by using antibodies against CD80 (eBioscience Inc.) for 1 h at 37 °C without permeabilization. Then, cells were washed with PBS 1X and slides were mounted and analysed with a confocal laser scanning microscope (Nikon A1R-A1). Image analysis was performed using the Nikon A1R-A1 software. Other immunofluorescence assays were performed by using antibodies against histone H2A.X (Genetex Inc.) and NF-kB p65 (Abcam) for overnight treatment at 4 °C after permeabilization. DRAQ5TM (Thermo Fisher) fluorescent probe solution was used to identify nuclei.
To test the presence of ROS, living cells were stained with 5 μM MitoSOX [3,8-phenanthridinediamine, 5-(6′-triphenylphosphoniumhexyl)-5,6 dihydro-6- phenyl] or 5 μM CM-H2DCFDA (all purchesad from Molecular Probes, Invitrogen, Carlsbad, CA) for 30 mins in CO2 incubator at 37 °C, after 30 mins and overnight treatment with pro-oxidants. For all these experiments, slides were mounted and analyzed with a confocal laser scanning microscope (Nikon A1R-A1 or Leica SP2). Image analysis was performed using the Nikon A1R-A1 software or Leica SP2 software.
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8

Cardiac Apoptosis Detection in Mice

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At the end of the treatment period, all mice were euthanized with an overdose of sodium pentobarbital (200 mg/kg, i.p.). Animal death was verified by observation of cardiac arrest and pupil enlargement for 1 min. The humane endpoints established in this study were as follows: Impaired ambulation that prevented animals from reaching food or water; excessive weight loss and extreme emaciation; lack of physical or mental alertness; difficult labored breathing; and prolonged inability to remain upright. Animals were observed a minimum of twice daily, with more frequent observations immediately after dosing and when increased morbidity or mortality was expected (17 (link)). The heart was then removed and fixed in 10% formalin for 24–48 h at 4°C. Formalin-fixed heart tissues were then embedded in paraffin and sections were cut (~4 µm thick). Cardiomyocyte apoptosis was detected using a one-step TUNEL Apoptosis assay kit at 37°C for 1 h (Roche Diagnostics GmbH), according to the manufacturer's protocol, followed by DAPI staining (10 µg/ml; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 2 min. Anti-fade mounting medium was then added (cat. no. P0126; Beyotime Institute of Biotechnology) to each slide. Images were obtained from five fields per slide using confocal microscopy (magnification ×400; NIKON A1R/A1; Nikon Corporation).
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9

Melatonin-Mediated Cytoskeleton Dynamics

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GBC-SD cells were seeded into 24-well plates with carry sheet glass and treated with melatonin (2 mM) for 6 h. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were re-washed 3 times with PBS and YE Dye phalloidin conjugates (US Everbright) staining was performed according to the manufacturer's instructions. DAPI was used for nuclear staining at room temperature for 5 min. The images were acquired using a laser scanning confocal microscope (Nikon A1R/A1; Nikon Corporation) (magnification ×400).
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10

Immunofluorescence Staining of Cells and DRGs

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Cells and DRGs were plated on 24-well chamber slides and allowed to attach overnight. Following drug treatment, the cells and DRGs were fixed in 4% paraformaldehyde for 30 min and then washed with PBS 3 times. Next, the samples were blocked in 5% bovine serum albumin (Sigma-Aldrich, Germany) for 1 h and then incubated with the primary antibody overnight. The samples were washed with PBS 3 times, and corresponding fluorescent secondary antibodies (Alexa Fluor 488 and 594, 1:2000, Thermo Fisher Scientific) were added for 1 h at room temperature in the dark. Nuclei were stained with DAPI (1:5000) for 15 min in the dark. Cells and DRGs were imaged using laser scanning confocal microscopy (Nikon A1R/A1).
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