For the in vivo experiment, the gastrointestinal system (stomach, duodenum, jejunum, ileum and colon) was freshly isolated. Then, intestinal segments rich in chyme were cut into 10 μm sections. Additionally, the intestinal contents of the mice were separated to make chyme smears that were stained with Bodipy or Oil Red-O. For the in vitro experiment, an appropriate amount of distilled water was added into DLA-M, HFF and HFF with DLA-M to obtain smears that were then stained with Bodipy or Oil-red O. Neutral lipids were visualized as green fluorescence at an absorbance wavelength of 488 nm using a fluorescence microscope (ZEISS Imager M1, Germany). Sections and smears were collected after lipid observation and were examined using a ZEISS Imager M1 polarized microscope to detect DLA-M crystals in situ36 (link). 3T3-L1 mature adipocyte-conditioned medium was collected as previously37 (link) and the content of the intestinal chime was separated as described above to mimic the physiological conditions of the intestinal environment for quantitative analysis.
Imager m1
Manufactured by Zeiss
Sourced in Germany
The Imager M1 is a high-performance microscope imaging system designed for advanced imaging applications. It features a modular design that allows for the integration of various imaging technologies, including brightfield, darkfield, and fluorescence microscopy. The Imager M1 is capable of capturing high-resolution images and videos, making it a versatile tool for a wide range of research and industrial applications.
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Lab products found in correlation
Axiocam mrm, by Zeiss (4 mentions)
Axiovision, by Zeiss (4 mentions)
Axiocam, by Zeiss (3 mentions)
Axiocam hrc, by Zeiss (3 mentions)
Axiovision 4, by Zeiss (2 mentions)
Fluorescent beads, by Polysciences (2 mentions)
Plan apochromat, by Zeiss (2 mentions)
Axiocam hrm camera, by Zeiss (2 mentions)
Tcs sp5, by Leica (2 mentions)
Axiocam mrc, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Alexa fluor 488 goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti actin antibody, by Merck Group (1 mentions)
Aec substrate chromogen, by Agilent Technologies (1 mentions)
Anti α smooth muscle actin α sma, by Merck Group (1 mentions)
Anti von willebrand factor, by Merck Group (1 mentions)
Haematoxylin, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
60 protocols using imager m1
1
In vivo and in vitro lipid analysis
2
Visualizing CaSR Expression in Zebrafish Larvae
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For immunocytochemical staining of the CaSR, zebrafish larvae were first fixed with 4% paraformaldehyde in PBS for 2 h at 4°C. After fixation, the larvae were briefly rinsed with PBS and were then gradually dehydrated with 100% methanol. Following rehydration with PBS, the larvae were blocked with 3% bovine serum albumin for 1 h. The larvae were then incubated with an rabbit anti-CaSR polyclonal antibody (1:500; Herberger and Loretz, 2013 (link); Kwong et al., 2014 (link)) or a rabbit anti-S100 polyclonal antibody (1:250; Dako, Carpinteria, CA, United States) in PBS at 4°C overnight. Subsequently, the larvae were washed with PBS for 20 min and incubated with an Alexa Fluor 488 goat anti-rabbit antibody (1:200; Invitrogen) for 2 h in the dark at room temperature. For double immunocytochemical staining, the larvae were incubated overnight at 4°C with CaSR polyclonal and mouse anti-actin monoclonal antibodies (1:100; Chemicon, Temecula, CA, United States). After washing, samples were incubated with Alexa Fluor 568 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit antibodies for 2 h at room temperature. Images were acquired using an upright microscope (Imager M1, Carl Zeiss, Oberkochen, Germany) or a Leica TCS-SP5 confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany).
3
Quantifying Pulmonary Arterial Remodeling
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Left upper lung tissues were fixed with 4% paraformaldehyde, paraffin-embedded, and sliced with 4 µm thickness. Lung tissue sections were stained with hematoxylin and eosin (H&E). To determine the extent of collagen deposition in pulmonary arteries, Sirius red staining and Masson trichrome staining was performed and quantified by modified ashcroft scoring system [15 (link)16 (link)]. For immunohistochemisty, the tissue sections were blocked with peroxidase blocking agent for 5 min, washed with Tris-HCl with Tween (TBST), and blocked with protein blocking serum free buffer for 5 min. Then, sections were incubated with anti-α-smooth muscle actin (α-SMA) (Sigma-Aldrich Co.) or anti-von Willebrand Factor (vWF) (Millipore, Temecula, CA) overnight at 4℃ followed by washing with TBST for 5 min. Enough labelled polymer conjugated with secondary antibodies (Dako, Carpinteria, CA) were applied to the slides for 30 min, followed by washing with TBST for 5 min. Peroxidase activity was detected with the ready-to-use AEC+substrate chromogen (Dako). At least twenty arteries of 15-100 µm per each rat were evaluated in α-SMA stained slides through a light microscope (imager M1, Carl Zeiss, Jena, Germany) at ×400 magnification and quantified by image J software.
The medial wall thickness is calculated as follows:
Wall thickness=(total area of artery–lumen area of artery)/total area of artery
The medial wall thickness is calculated as follows:
Wall thickness=(total area of artery–lumen area of artery)/total area of artery
4
Evaluating Gastrointestinal Responses to Lipids
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For the in vivo experiment, RFP mice were treated as described above, and the gastrointestinal contents (stomach, duodenum, jejunum, ileum, cecum and colon) were evaluated by using smears. BODIPY® FL C16 and FITC-LPS were visualized as green fluorescence at an absorbance wavelength of 488 nm using a fluorescence microscope (ZEISS Imager M1, Germany), and DLA-M crystals were detected using a polarized brightfield in situ. For the in vitro experiment, the intestinal chyme was diluted with saline or certain concentrations of OA, PA and LPS and then treated with 0–400 mg/ml DLA-M, laumontite and maifanite for 6 h. Afterward, the supernatant concentration was measured.
5
Immunohistochemical Analysis of VEGF-A in Temporal Artery Biopsies
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Paraffin-embedded temporal artery samples were obtained from the TABUL study for five patients; three had a positive biopsy and two were control patients. Sections were cut at 4 μm and mounted on adhesive glass slides for staining. Slides were deparaffinised in xylene and rehydrated through a graded series of 100–50% ethanol. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide. Heat-induced antigen retrieval for VEGF-A was performed by citrate buffer (10 mM anhydrous citric acid, 0.05% Tween 20, pH 6.0). Non-specific reactivity was blocked in buffer solution containing 3% filtered bovine serum albumin (BSA). Representative sections from each patient were incubated with 1:100 dilution of rabbit anti-human VEGF polyclonal IgG antibody (ABCAM). Negative control sections were incubated with non-immune rabbit IgG (R&D Systems). Secondary biotinylated goat anti-rabbit antibody (Vector Laboratories) was added at 1:250 dilution before incubation with avidin-biotin-peroxidase (ABC, Vectastain Elite kit, Vector Laboratories). Staining was developed with DAB substrate kit (Vector Laboratories) and counterstained with haematoxylin (Vector Laboratories). Images were captured using a microscope (Zeiss Imager M1) connected to a camera (Zeiss Axiocam). For general morphological analysis, serial sections were stained with Mayer's H&E.
6
Twist1 Protein Immunostaining and Cartilage/Bone Staining
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Staining for Twist1 protein was carried out on frozen sections of E11.5 forelimb buds using mouse monoclonal anti-Twist1 antibody 2C1A (Abcam ab50887, 1/50 dilution) and AlexaFluor 488 conjugated donkey anti-mouse secondary antibody (Life Technologies) as described [15] (link). Stained sections were imaged on a Zeiss Axio Imager M1. For bone and cartilage staining, embryos were collected in water at E17.5, skinned and stained as whole specimens as described previously [15] (link), [19] . Specimens were stored in 80% glycerol and limbs were photographed with a Leica microscope and SPOT digital camera.
7
Indirect Immunocytochemistry of Gonads
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Indirect immunocytochemistry of extruded and 1% paraformaldehyde PFA-fixed gonads was carried out in solution as described (23 (link)). Images were taken on a Zeiss Imager M1 equipped with an Axiocam MRm (Zeiss) and processed with AxioVision (Zeiss) and Photoshop CS5 (Adobe). Secondary antibodies coupled to fluorochromes FITC, CY3 and CY5 were purchased from Jackson ImmunoResearch (Dianova).
8
Quantifying Endogenous ROS Levels in C. elegans
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Endogenous ROS levels were measured using a ROS-sensitive probe H2DCFDA.41 (link),42 (link) Synchronized worms were collected and washed with M9 buffer. Worms were then incubated in M9 supplemented with 50 μM H2DCFDA for 2 h at 37°C. Finally, they were observed under a Zeiss Imager M1 with a AF488 filter. Fluorescence intensity of individual worms was quantified using the ImageJ software.
9
Fluorescence Imaging of PEX Proteins
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Cells expressing PEX26 or PEX15 constructs were analyzed by direct fluorescence and immunofluorescence experiments. If not indicated otherwise, cells were transfected for 48 hours using Effectene (Qiagen). Fixation, permeabilization, incubation with antibodies, and mounting was carried out as described58 (link)59 (link). The following antibodies were used: anti-PEX14 (ProteinTech, rabbit polyclonal, dilution 1:200) and anti-Myc (Cell Signaling, mouse monoclonal, dilution 1:2000), anti-Catalase (Oxis International, rabbit polyclonal, 1:1000), anti-mouse Alexa488 (MoBiTech, dilution 1:200), and anti-rabbit Cy3 (Jackson ImmunoResearch, dilution 1:200). Yeast cells were mounted in low-melting agarose for imaging. Samples were analyzed using a 100x oil immersion objective (NA 1.3) on a Zeiss Imager M1 fluorescence wide field microscope with a HRm Camera and the Axiovision 4.8 acquisition software. For yeast microscopy widefield images were contrast-enhanced and false-colored in blue to show cell contours.
10
Comprehensive Aquatic Ecosystem Analysis
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HRT was calculated using discharge rates and the volume provided by K-water (http://kwater.or.kr ). BOD5 was measured by standard methods [23 ], and CODMn was analyzed by estimating the consumption of potassium permanganate. Chl-a was analyzed using a spectrophotometer (Lambda 45; PerkinElmer, Waltham, MA, USA), for which the water samples were filtered through Whatman GF-F filters and extracted with 90% acetone for 24 h [23 ]. TN and TP contents were analyzed using an autoanalyzer (INTEGRAL Futura; Ams Alliance, Frépillon, France). The NO3−-N content was analyzed using ion chromatography (850 Professional IC; Metrohm, Switzerland). Concentrations of NH4+-N and PO43−-P were determined with an ion analyzer (Smartchem 140; Ams Alliance, Frépillon, France). Phytoplankton samples were identified to the genus or species level following John et al. [24 ] and Komárek and Anagnostidis [25 ,26 ]. For quantitative analysis, 1 mL of a sample was added to and allowed to settle in a Sedgwick–Rafter counting chamber, followed by examination under an optical microscope (Imager M1; Carl Zeiss, Oberkochen, Germany) at a 200× to 400× magnification to calculate the cell density per milliliter.
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