To record high-resolution 4D (XYZT) time-lapse stacks, worms mounted with the slide-and-oil method were imaged with DIC optics on a Zeiss Axioplan2 microscope equipped with a Zeiss AxioCam HRc camera using the 6D Acquisition module of AxioVision v4.8.2 (Zeiss, Oberkochen, Germany). We used a custom-made stage micrometer to calibrate images, enabling us to derive absolute measurements from them. Z-stacks comprising focal planes spaced 3 μm apart were acquired every 2 min. Imaging captured the wound site and up to four adjacent segments. Movies of selected focal planes were generated using AxioVision.
Axiovision v4
Manufactured by Zeiss
Sourced in Germany, United States, Japan, Switzerland
AxioVision v4.8 is a software package designed for microscope image acquisition, processing, and analysis. It provides a comprehensive set of tools for researchers and scientists working with Zeiss microscopes. The software enables users to capture, manage, and analyze high-quality digital images from a variety of Zeiss microscope systems.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam mrc5, by Zeiss (9 mentions)
Axio observer z1, by Zeiss (6 mentions)
Axiocam mrm, by Zeiss (5 mentions)
Axiocam hrc camera, by Zeiss (4 mentions)
Axiovert 200 microscope, by Zeiss (4 mentions)
Lsm 510 meta, by Zeiss (4 mentions)
Triton x 100, by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Axiovert 200m microscope, by Zeiss (4 mentions)
Axiocam mrc, by Zeiss (4 mentions)
Axiovert 200m, by Zeiss (4 mentions)
Axioscope, by Zeiss (4 mentions)
Axioplan 2 microscope, by Zeiss (3 mentions)
Axiocam mrc camera, by Zeiss (3 mentions)
Plan apochromat, by Zeiss (3 mentions)
Axioskop2 mot plus microscope, by Zeiss (3 mentions)
Axioskop 2 plus, by Zeiss (3 mentions)
Prolong antifade kit, by Thermo Fisher Scientific (3 mentions)
Observer z1, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
106 protocols using axiovision v4
1
High-Resolution 4D Time-Lapse Imaging of Worms
2
Adipocyte Size Quantification in Adipose Tissue
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For the determination of adipocyte size, paraffin-embedded eWAT tissues were sectioned at 5 µm using a Reichert microtome (Reichert-Jung 2030, Cambridge Instruments, Heidelberg, Germany) and mounted on Superfrost plus slides (Menzel-Gläser, Menzel GmbH & Co. KG, Braunschweig, Germany). Sections were stained with PAS-Haematoxilin, resulting in blue-colored nuclei and purple cell membranes and cytoplasm. Tissues were examined under an Axioskop 2 microscope, and jpg images were acquired at 20× magnification using an Axiocam MR5 camera and AxioVision software v4.82 (Zeiss GmbH, Jena, Germany). Average adipocyte size was determined on digital images using AxioVision v4.82 (Zeiss GmbH, Jena, Germany), by drawing cell circumferences and calculating surface area in µm2 of 400 adipocytes per animal.
3
Testicular Morphometry and Spermatogenesis
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PG1, PG2, PN1, and H1 underwent unilateral orchiectomy after electroejaculation procedure. PG3, PN2, and H2 had their testicles collected immediately post-mortem. Testicular tissue was grossed into 1 cm thick sections, fixed in Bouin’s fixative for 24 h, processed for paraffin embedding, microtome-sectioned at 5-μm thickness, stained with hematoxylin and eosin, and imaged with a microscope. Then, 60 round or nearly round tubular profiles from each animal were randomly chosen and had diameter and epithelium height measured (Axio Vision v. 4.8.2, Carl Zeiss AG, Feldbach, Switzerland) size measurement tools were used). Ten sections of seminiferous tubules were analyzed to quantify the population of sperm cells. The results were presented with mean ± SD.
4
Quantifying Lung Inflammation and Mucus Production
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Recruitment of inflammatory cells into the lungs and morphological alterations of the tissue were assessed after H&E staining. The number of mucus-producing goblet cells was determined after PAS-staining from three bronchi per mouse in control group and six bronchi per mouse in treatment groups by counting PAS+ cells from 200 μm of bronchus surface under light microscope.
Quantification of histological alterations was performed by measuring all inflammatory areas and areas including nuclear dust of one lung section per slide at a total magnification of 100x with AxioVision V4.8.2 software (Zeiss, Oberkochen, Germany). The results were expressed as averages of the measured areas.
Quantification of histological alterations was performed by measuring all inflammatory areas and areas including nuclear dust of one lung section per slide at a total magnification of 100x with AxioVision V4.8.2 software (Zeiss, Oberkochen, Germany). The results were expressed as averages of the measured areas.
5
Immunofluorescence Staining for Cell Typing
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Indirect immunofluorescence was carried out on cells grown on ECM-coated coverslips. For fibroblasts, positive staining was for Vimentin; for epithelial cultures, staining was for a specific cytokeratin. Cells were then imaged on a Zeiss Axioplan2 using a 63× / 1.40 Plan Apochromat objective and analysed with Axiovision v4.8.2 (Zeiss). Specific band pass filter sets for DAPI, FITC and Cy5 were used to prevent bleed through. Images were processed using Fiji ImageJ. Some data were generated with University of Manchester software; https://github.com/zindy/libatrous . Antibodies against the listed proteins were used as follows: Vimentin (diluted 1:1000, Santa Cruz, cat. no. sc-7557), pan-cytokeratin (diluted 1:1000, Abcam, cat. no. Ab27988), cytokeratin 5 (diluted 1:2000, Covance, cat. no. PRB-160P), cytokeratin 14 (diluted 1:1000, Covance, cat. no. PRB-155P), cytokeratin 8/18 (diluted 1:200, Progen, cat. no. Gp11) and cytokeratin 19 (diluted 1:10, generated in-house). Antibodies were assessed for specificity by western blotting. All antibodies detected bands only at the expected size.
6
Quantifying Pulmonary Neutrophil Infiltration
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PMN detection in the lung tissue was performed by using a Vectastain ABC kit (Vector Laboratories; Linaris; Dossenheim; Germany). Sections were blocked with avidin solution (Vector Laboratories; Burlingame, CA, USA) for one hour to avoid unspecific binding sites and followed by incubation with a rat anti-mouse Ly6G antibody (clone RB6-8C5; ab25377; Abcam; Cambridge, UK). Sections were incubated with biotinylated anti-rabbit IgG (BA-4000; Vector Laboratories; Germany) for one hour, followed by Vectastain ABC reagent (PK-4000; Vector Laboratories; Germany) for 30 min and then incubated with DAB substrate. Nuclear fast red (Linaris; Germany) was used for tissue counterstaining. Rat IgG was utilized as a control (sc-2026; Santa Cruz Biotechnology; Dallas, TX, USA). Tissue slides were processed with a Leitz DM IRB microscope (Leica; Wetzlar, Germany) and analyzed with AxioVision v4.8.2 (Zeiss microscopy; Jena, Germany). PMN counts were enumerated from four random sections in each group by independent observers and reported as number of PMNs per high power field (HPF) as described before [40 (link)].
7
Karyological Analysis of Luzula Species
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For the karyological analyses, we collected seeds from 13 populations of L. alpina, L. exspectata and L. multiflora in the Eastern Alps and one of L. taurica from the central Balkan Peninsula during field excursions between 2016 and 2021 (Table S1 , Figure 3 ). Seeds were germinated and root tips fixed, Feulgen-stained and squashed to prepare microscopic slides as described by Bačič et al. [28 (link)]. The chromosome number was determined using an Axioscope MOT light microscope (Carl Zeiss, Jena, Germany) with a 63× oil immersion objective, CCD camera (Sony DXC-950P; Sony, Köln, Germany), frame grabber Matrox Meteor (Matrox, Dorval, Quebec, Canada) and computer with the image analysis software KS 400 v. 3.0 (Carl Zeiss, Jena, Germany). Photographs of the chromosomes were produced using AxioVision v. 4.8.2 (Carl Zeiss, Jena, Germany).
8
Microscopic Analysis of Cellular Samples
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Microscopical analyses were performed by Zeiss Axioplan 2 microscope, as described by Erdelyi and co [12 (link)]. For brightfield pictures DIC filter, for fluorescence images the appropriate fluorescence filter was used. All images were acquired with a Zeiss AxioCam color camera driven by AxioVision v4.8.2 software (Carl Zeiss Microscopy, Jena, Germany). Images were adjusted for contrast, cropped, and merged using Adobe Photoshop.
9
Quantifying Neutrophil Accumulation in Tissues
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PMN accumulation in peritoneal tissue, lung and liver sections was visualized via immunohistochemistry by using a Vectastain ABC kit (PK-4000; Vector Laboratories; Germany). Sections were blocked with Avidin solution (Vector Laboratories; Germany) for 1 h to avoid unspecific binding sites. PMNs were stained with rabbit anti-mouse Ly-6G (clone 1A8; Abcam; UK). Rabbit IgG was used as control (31235; Invitrogen; USA). Sections were incubated with biotinylated anti-rabbit IgG (BA-4000; Vector Laboratories; USA) for 1 h, followed by Vectastain ABC reagent (PK-4000; Vector Laboratories; USA) for 30 min and then incubated with DAB substrate. Nuclear fast red (H-3403; Linaris; Germany) was used for tissue counterstaining. Tissue slides were processed with a Leitz DM IRB microscope (Leica) and analyzed with AxioVision v4.8.2 (Carl Zeiss MicroImaging; Germany). Neutrophil counts were examined by enumerating the positive and therefore brown stained cells in a masked fashion. PMN numbers were scored from four random sections of four different tissue samples in each group by two independent observers (41 (link)).
10
Wood Anatomy Quantification Protocol
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For all species, thin (20 μm) traverse sections of the wood anatomical samples were made with a sliding microtome (G.S.L.1; Schenkung Dapples, Zürich, Switzerland), stained with safranin-alcian blue, rinsed with distilled water and ethanol (95%) and permanently embedded on glass slides using Euparal (Carl Roth, Karlsruhe, Germany). Subsequently, the complete cross-section was digitalized at 100-times magnification using a light microscope equipped with an automated table and a digital camera (Observer.Z1 and Software: AXIOVision v.4.8.2; Carl Zeiss MicroImaging GmbH, Jena, Germany). Anatomical measurements were made by semi-automated image analysis using IMAGEJ v.1.52p (Schneider et al., 2012) and GIMP v.2.10.6 (GIMP Development Team, 2018) . While the majority of vessels could be identified by applying this semi-automated approach, it was impossible to completely exclude tracheids in our measurements of some species because narrow vessels cannot be easily distinguished from tracheids based on transverse sections. The equivalent vessel diameter according to White (1991; d, μm) , that is the diameter of a circular vessel with the same conductivity as an elliptical one with minor and major radius a and b, was calculated as:
Based on d, the hydraulically weighted average vessel diameter (D h , μm) was calculated according to Sperry et al. (1994) as:
Eqn 2
Based on d, the hydraulically weighted average vessel diameter (D h , μm) was calculated according to Sperry et al. (1994) as:
Eqn 2
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