To determine injection placement, GFP was imaged with a Zeiss Axio Imager Z2 microscope using the 10x objective. To examine GFP co-localization with vGluT1, digital images were captured with optical sectioning (63x objective) to permit co-localization within a given z-plane (0.5-μm thickness). Co-localization was defined as yellow fluorescence from the overlap between labeled GFP terminals and red-colored vGluT1. The Zeiss Axio Imager Z2 microscope was also used to collect tiled brightfield images (10x objective) of FosB/ΔFosB-labeled tissue sections bilaterally. Imaging of dual-fluorescent FosB/ΔFosB and CaMKIIα or GAD67 was carried out with a 63x objective and optical sectioning (0.5-μm thickness) to determine if nuclear FosB/ΔFosB was surrounded by cytosolic markers for glutamatergic and/or GABAergic neurons within a 0.5-μm z-plane. SynaptoTag-injected tissue was imaged with the 10x objective to verify mCherry expression in the IL and GFP labeling in the insula. High-magnification images of GFP appositions onto CaMKIIα- or GAD67-labeled cell bodies in the insula were captured with a 63x objective using 0.5-μm thick optical sectioning. In addition to GFP and Cy3 CaMKIIα or GAD67, a Cy5 image was acquired and subtracted during processing to ensure exclusion of potential off-target or autofluorescent signals.
Axio imager z2 microscope
Manufactured by Zeiss
Sourced in Germany, Canada, United States, United Kingdom
The Axio Imager Z2 is a high-performance microscope designed for advanced imaging and analysis. It features a modular design that allows for customization to suit various research and laboratory needs. The microscope offers excellent optical performance, precise control, and a range of imaging techniques to support a wide variety of applications.
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Lab products found in correlation
Axiocam 506, by Zeiss (9 mentions)
Zen blue software, by Zeiss (8 mentions)
Axiocam mrc, by Zeiss (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
Zen 2, by Zeiss (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
Isis software, by MetaSystems (6 mentions)
Axiocam mrm, by Zeiss (6 mentions)
Tissuefaxs plus system, by Zeiss (6 mentions)
Levamisole, by Merck Group (5 mentions)
Tissuefax, by TissueGnostics (5 mentions)
Axioplan 2 microscope, by Zeiss (5 mentions)
Ld lci plan apochromat 63x 1.2 imm corr, by Zeiss (5 mentions)
Metafer4, by MetaSystems (5 mentions)
Cool cube 1 camera, by MetaSystems (5 mentions)
Axiocam mr r3, by Zeiss (4 mentions)
Zen 3, by Zeiss (4 mentions)
Permount, by Thermo Fisher Scientific (4 mentions)
Stereo discovery v12, by Zeiss (4 mentions)
Cm1900, by Leica (4 mentions)
326 protocols using axio imager z2 microscope
1
Multimodal Imaging of Neuronal Markers
2
Comprehensive Bone Tissue Analysis Protocol
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After micro-CT analysis, femurs were included in methyl methacrylate (MMA) at 4°C. Serial 5μm sections were cut in three distinct levels, eight sections per level, with a 50μm interval between levels. Relative osteoid surface (Osteoid / Bone surfaces = OS/BS *100%) and number of osteoblast per bone surface N.Ob/BS (mm-1) were determined in 2 sections per level in each animal by staining with toluidine blue (1%) as described previously [19 (link)]. The Axioimager Z2 microscope (Zeiss, Germany) was used for osteoid and bone surfaces identification and each area was measured with Axiovision software (Zeiss, Germany) at 200x magnification. Bone and osteoid surfaces were measured in the distal metaphysis of the femur. To determine the osteoblasts number (N.Ob) in OS a magnification of 400x was used (Axioimager Z2 microscope, Zeiss, Germany). Osteoclasts were stained with naphthol AS-TR (3- hydroxy-2-naphthoic acid 4-chloro-2-methylanilide) phosphate for tartrate-resistant acid phosphatase (TRAP) detection and counterstained with Toluidine blue (0.5%), [20 (link)]. Osteoclasts were counted and expressed relatively to bone surface as Oc.S/B.Ar (mm2). To evaluate total iron accumulation in femur trabecular bone, sections were stained with Perl’s solution ((2% Ferrocyanide potassium) 1:1 (2% HCl (37%)) and counterstained with Nuclear Fast Red (1%).
3
Spinal Cord Cryosectioning and Imaging
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The spinal cord segments from C3 to T3 were dissected, cryoprotected in 30% sucrose in 0.1 M PBS pH 7.4 for 48 h and embedded in OCT compound (ProSciTech Pty Ltd, QLD, Australia). Spinal cord sections were cut transversely at 10 µm on a cryostat (Leica CM 1950 Cryostat, Amtzell, Germany). Sections were thawed in a 37 °C oven for 10 min, then washed twice for 10 min in Tris phosphate buffered saline (TPBS = 0.05 M PBS + 0.01 M TRIS). The sections were then treated with 50% ethanol/TPBS for 20 min, followed by three 10 min washes in TPBS. DAPI (1 µg/mL) was applied to each slide, incubated for 1 min to visualize the cell nuclei and then washed twice for 10 min. The sections were then coverslipped with fluorescence mounting medium (DAKO, S3023, Carpinteria, CA, US). The sections were imaged with a Zeiss Axio Imager Z2 microscope (Carl Zeiss Microimaging GmbH, Germany). Images were acquired from C3 to T3 for quantitative image analysis. All images were taken at 20× magnification and exposure times were kept constant.
4
BrdU-based Proliferation Assay in Bone and Intestinal Tissues
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To assess cell proliferation, frozen bone sections were rehydrated in PBS, and paraffin ileum sections were deparaffinized and rehydrated in graded alcohol. A 1% trypsin in PBS solution was placed on the tissue for 10 min at 37°C. Slides were rinsed in PBS followed by 4 N HCl for 30 min at room temperature. Slides were rinsed in PBS then blocked in mouse‐on‐mouse blocking reagent (Vector Labs, Burlingame, CA, USA) for 10 min. A biotinylated monoclonal BrdU antibody (B35138, diluted 1:50 in PBS; Thermo Fisher Scientific, Waltham, MA, USA) was placed on the tissue and incubated overnight at 4°C. Frozen sections were brought to room temperature, rinsed in 1X PBS, and incubated with a Alexa Fluor 488 Streptavidin conjugate (S11223, diluted 1:100 in PBS; Thermo Fisher Scientific) for 1.5 hours. Sections were then rinsed and cover‐slipped with Prolong Diamond Antifade with DAPI (P36966; Thermo Fisher Scientific). Following antibody treatment, paraffin ileum sections proceeded with a HRP‐streptavidin and DAB substrate reaction and were counterstained with hematoxylin. For paraffin histology, bright‐field BrdU images were obtained using the Zeiss Axio Imager Z2 microscope and a 20× objective. For frozen histology, fluorescent images were obtained along the periosteal compressive surface on the Zeiss Axio Imager Z2 with the GFP filter used to detect BrdU Alexa 488.
5
Caryopsis Histological Preparation and Visualization
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Caryopsis were collected and fixed in FAA (50% ethanol:formalin:acetic = 89:5:6, v/v) and then vacuum-infiltrated for 30 min, replacing the solution with new FAA or 70% ethanol and incubating overnight. Dehydrated in an ethanol series (30%, 50%, 70%, 80%, 90%, and 100%) and then washed twice in absolute ethanol for 30 min. Embedded in melted Paraplast (Sigma, USA) and sectioned to a thickness of 8 μm with a microtome (Leica Microsystems RM2145). After dewaxing with TO transparent agent, the sections were hydrated through an ethanol series (100%, 90%, 70%, 80%, 50%, and 30%) and then washed twice in distilled water for 30 min, stained with 1% toluidine blue for 45 s, then sealed with resin. Sections were visualized under a Zeiss Axio Imager Z2 microscope (Carl Zeiss, Germany).
6
Exosome-Mediated Proliferation of hCECs
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hCECs were seeded in 24-well plates on coverslip glasses at 2 × 104 cells/well in complete DH medium. At 6 h post-seeding, different types of exosomes (800 μg) or vehicle alone (HBS; negative control) were added to cultures. Cells were incubated for 48 h at 37 °C before they were fixed in 4% formaldehyde. Cells were then permeabilized with 0.2% Triton X-100 for 10 min and incubated with the following primary antibody: mouse monoclonal antibody against Ki-67 (1:200, #556003, BD Biosciences, Franklin Lakes, NJ, USA). Samples were washed with PBS before addition of secondary antibody, peroxidase-conjugated AffiniPure Goat anti-mouse IgG488 (1:400, A11059, Invitrogen). All antibodies were diluted in PBS containing 1% bovine serum albumin. Cell nuclei were counterstained with Hoechst reagent 33258 (1:100; Sigma). Coverslips were mounted on glass slides with mounting medium and kept at 4 °C until observation with an epifluorescence microscope (Zeiss Axio Imager Z2 microscope; Zeiss Canada Ltd.). Samples were photographed with a numeric CCD camera (AxioCam MRm; Zeiss Canada Ltd.). Negligible background was observed for controls (primary antibodies omitted). Number of Ki-67-positive cells was counted and expressed as a percentage of the total number of cells.
7
Neuron Morphometry Using Microscopy and Image Analysis
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After patch-clamp recording and filling with biocytin/neurobiotin tracer, horizontal midbrain slices were fixed in 4% paraformaldehyde (PFA) solution for 24 hr at 4°C. All following staining procedures were done as described previously (Marx et al., 2012 (link); Mao et al., 2019 (link)), depending on the tracer. Biocytin-filled, DAB (3,3′-Diaminobenzidine) stained neurons (N = 27) were imaged with Zeiss Axio Imager Z2 microscope (Zeiss AG, Oberkochen, Germany) in transmitted light mode. Neurobiotin-filled, Streptavidin-Alexa 633 stained neurons (n = 22) were imaged with multiphoton Zeiss LSM 7 MP system. 3D morphology reconstruction was done with Simple Neurite Tracer plugin (https://imagej.net/Simple_Neurite_Tracer ) in Image J software, followed by inbuilt Sholl analysis instrument.
8
Immunofluorescence Analysis of mESCs
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Undifferentiated or differentiated mESCs were fixed in 4% paraformaldehyde, incubated with primary antibodies (diluted in 1x PBS, 1% Bovine Serum Albumin, 0.05% sodium azide solution) overnight at 4°C, listed in Supplementary Table 2 , followed by appropriated secondary antibody incubation, overnight at 4°C. Nuclei were stained with DAPI at room temperature and cell images were acquired with Zeiss Axio Imager Z2 microscope or Zeiss LSM710 confocal microscope (Carl Zeiss). Images were taken in sequential mode and posteriorly adjusted in ImageJ.
9
Quantitative Morphometry of Sympathetic Axons
10
Histological Evaluation of Skeletal Muscle Atrophy
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All histological experiments were completed as previously described by us [36 (link),105 (link)]. To determine whether IRI had atrophic effects on skeletal muscle and, subsequently, whether BGP-15 adjuvant therapy could rescue any atrophy, we histologically assessed TA muscles which were cryopreserved in optimal cutting temperature compound (Sakura Finetek, Maumee, OH, USA). TAs were sectioned (10 µm, −20°C, Leica CM1950, Leica Biosystems, Mount Waverley, Australia) and mounted onto glass slides and then stained with hematoxylin and eosin (H&E) to evaluate muscle fiber size through CSA. All slides were imaged on a Zeiss Axio Imager Z2 microscope (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany) at 20× magnification and analyzed as described previously [36 (link),105 (link)] using ImageJ software (NIH, Bethesda, MD, USA).
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