WISH, SISH, histological stainings and IHC on sections of all mouse experiments were documented with a Leica DM5000 microscope equipped with a Leica DFC300FX digital camera and the used software was Leica FireCam (Version 1.9.1). Immunofluorescence staining of mouse tissue sections were photographed with the Leica DMI6000 microscope equipped with a Leica DFC350FXR2 camera and the Leica Application Suite X software. Images were processed in Adobe Photoshop CS4 or CC, or FIJI ImageJ version 2.0.0, figures assembled using Adobe Illustrator CS4, CS6 or CC. Xenopus WISH was imaged with a Zeiss SteREO Discovery.V12 or if sections were made with a Zeiss Axioskop 2 mot plus each equipped with an AxioCam HRc in combination with AxioVision 4.7. Fluorescence imaging of Xenopus was performed on a Zeiss AxioObserver with a LSM700 and Zeiss ZEN black software. CBF was documented with a Zeiss Axioskop 2 mot plus equipped with a high-speed Hamamatsu video camera Orca flash 4.0 and Zeiss ZEN blue. CGF analysis was done with a Zeiss Axioskop 2 mot plus combined with a Zeiss AxioCam HSm camera and Zeiss AxioVision 4.7. CBF and CGF was assessed using the ImageJ plugin Particle Tracker56 (link). All trajectories were further analysed using a custom-made program written in RStudio which calculated the velocity of each particle track57 (link).
Axioskop 2 mot
Manufactured by Zeiss
Sourced in Germany
The Axioskop 2 mot is a high-performance microscope system designed for advanced microscopy applications. It features a motorized stand that enables smooth and precise movements, allowing for efficient sample observation and image capture. The Axioskop 2 mot is a versatile tool suitable for a wide range of microscopy techniques.
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Lab products found in correlation
Axiocam hrc, by Zeiss (3 mentions)
Axiocam mrm, by Zeiss (3 mentions)
Axio observer, by Zeiss (2 mentions)
Sybr green 1, by Merck Group (2 mentions)
Sonifier 2200, by Emerson (2 mentions)
Nuclepore black filters, by Cytiva (2 mentions)
Whatman, by Cytiva (2 mentions)
Axiovision software, by Zeiss (2 mentions)
Axiocam mrc5, by Zeiss (2 mentions)
Axiocam hr, by Zeiss (2 mentions)
Lsm 700, by Zeiss (1 mentions)
Dm5000 microscope, by Leica (1 mentions)
Dmi6000 microscope, by Leica (1 mentions)
Stereo discovery v12, by Zeiss (1 mentions)
Application suite x software, by Leica (1 mentions)
Orca flash 4, by Zeiss (1 mentions)
Axiocam hsm camera, by Zeiss (1 mentions)
Dfc300fx digital, by Leica (1 mentions)
C9100 13 emccd camera, by PerkinElmer (1 mentions)
Csu 10 spinning disk head, by Hamamatsu Photonics (1 mentions)
31 protocols using axioskop 2 mot
1
Microscopy and Image Analysis Methods
2
Worm Immobilization for Confocal Imaging
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Worms were immobilized with 40 mM levamisole or 0.1 μm diameter polystyrene microspheres in water (Polysciences 00876‐15, 2.5% w/v suspension) on 10% agarose inwater pads. Unless noted otherwise, imaging was performed on a spinning disc confocal system (WaveFX system from Quorum Technologies), consisting of an inverted Zeiss AxioObserver equipped with a 100× oil (N.A. 1.4) objective, Yokogawa CSU-10 spinning disk head, 491 & 561 nm lasers, and a Hamamatsu C9100‐13 EMCCD camera, controlled by Volocity software version 6.5.1 (PerkinElmer). Body-length imaging was completed on a Zeiss Axioskop 2 mot with a 100× oil (N.A. 1.3) objective, FluoArc/HBO103 illuminator, via a Hamamatsu Orca ER C4742‐80 CCD camera and Open Lab (V5) (Agilent) software.
3
Histological Analysis of Vascular Grafts
4
Invadopodia Assay Protocol
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Invadopodia assays were performed following the manufacturer’s instructions (ECM670, Millipore, Zug, Switzerland). For the experiment, 1.2x103 cells were seeded onto FITC-labeled gelatin into 8-chamber slides and incubated at 37°C for 24h or 48h. Cells were fixed with 4% formaldehyde for 30 min, washed and stained with a solution of TRITC-phalloidin (2 μg/ml) and DAPI (1 μg/ml) in fluorescent staining buffer (PBS with 2% blocking serum and 0.25% Triton X-100) for 1h at RT, protected from light. Coverslips were mounted on the slides using hard set mounting medium (Reactolab) and pictures were taken at 20x magnification with a fluorescence microscope (Axioskop 2 Mot, Zeiss, Feldbach, Switzerland). Image analysis was performed using the program ImageJ [33 (link)].
5
Histological Neuron Reconstruction and Simulation
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After each successful recording, the pipette was slowly retracted from the cell, and the slice fixed in a 0.1 M phosphate buffer solution containing 4% paraformaldehyde and stored in the refrigerator. Histology was done based on previously published methods41 42 (link). Stained neurons were imaged on a standard microscope (AxioSkop 2MOT, Zeiss, Germany) equipped with a high-resolution color camera. Neurons were reconstructed using Neurolucida software (version 10; MBF Bioscience, Williston, USA) and exported to NEURON (ver 7.3) simulating environment43 for visualization.
6
Quantification of Senescence-Associated β-Galactosidase
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Staining of SA-ß-galactosidase (SA-ß-gal) in cells was carried out in triplicates using Histochemical Staining Kit (CS0030-1KT, Sigma-Aldrich) according to manufacturer´s instructions. Quantitative analyses were performed using an Axioskop 2 mot plus provided with a motorized stage (Zeiss, Oberkochen, Germany) and a CX 9000 digital camera (MicroBrightField Europe, Magdeburg, Germany) and the Stereo Investigator 8.1 software (MicroBrightField). Quantification was performed using the dissector method [9 (link)]. Cells of different regions of each sample were selected randomly for software supported counting.
7
Quantifying Prokaryotic Abundance in Sediments
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Total prokaryotic abundance was determined by epifluorescence microscopy47 (link). Sediment samples were treated three times for 1 min by ultrasounds (Branson Sonifier 2200, 60 W) after addition of 0.2 µm pre-filtered tetrasodium pyrophosphate solution at a final concentration of 5 mM, then properly diluted before filtration onto 0.2 µm pore-size Nuclepore black filters (Whatman). Each filter was then stained with 20 µl of SYBR Green I (Sigma Chemicals, previously diluted 1:20 with 0.2 µm pre-filtered Milli-Q water), washed twice with 3 ml sterilized Milli-Q water and mounted onto microscope slide. Filters were analyzed using epifluorescence microscopy (Zeiss Axioskop 2MOT, magnification 1,000×). At least 20 microscope fields and 400 cells were respectively observed and counted for each filter48 (link). Prokaryotic abundance was expressed as cells per g of dry sediment, after desiccation at 60 °C for 24 h45 . Prokaryotic biomass was determined based on cell size, converted into bio-volume, assuming 310 fg C m3 as a conversion factor, following standard inter-calibration with Scanning Electron Microscope (SEM)45 ,48 (link),49 .
8
Quantitative Analysis of Peripheral Nerve Myelination
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Analysis of axon caliber, myelination thickness and axon density, was conducted on sciatic nerve semithin sections removed from transcardially perfused mice. The perfusion solution contained 3% paraformaldehyde and 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Sections were postfixed for 1 h and kept in a fixative which included 3% sucrose. Images of toluidine blue-stained semithin sections were taken using an Axioskop 2 MOT (Carl Zeiss, Germany), equipped with a 100× immersion oil objective and an Olympus XC50 camera (Olympus, Germany). Standardized settings for camera sensitivity, resolution (2,576×1,932 pixels) and illumination were used for all micrographs. Image analysis was conducted with ImageJ version 1.43u. Employing freehand selection tool, axon and myelin were grossly circumscribed and the area adapted using the ABSnake plugin (the gradient threshold varied between 20 and 30, 10 to 20 iterations were used per image). Based on the areas measured, the thicknesses of the axons and myelin sheaths were calculated. Axons with distorted myelin sheaths were excluded from measurement as fixation artefacts. Axon density was only quantified with regard to myelinated axons.
9
Isolation and Analysis of Mouse GV Oocytes
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Germinal vesicle (GV) oocytes were obtained from 8 to 9 weeks old female Brg1fl/fl and Brg1fl/fl;Amhr2-Cre mice, 44–46 h post-eCG (5 IU i.p.) by placing the ovaries in PBS and using a 26-gage needle to puncture the ovaries. Only oocytes fully enclosed by cumulus cells were collected and washed in PBS with gentle agitation until denuded. Oocytes were immediately incubated in 4% paraformaldehyde for 20 min at 37°C, washed three times in PBS, and stained with Hoechst 33342 diluted at 1/300 in PBS for 15 min. Stained oocytes were mounted on slides using mounting media (Immu-mount, Thermofisher) and observed using the Zeiss Axioskop 2 MOT (Oberkochen, Germany) fluorescence imaging microscope at ×40 magnification. Assessment of GV chromatin configuration with non-surrounded nucleus (NSN) or surrounded nucleus (SN) was performed and reported as proportions of SN and NSN oocytes from a minimum of three mice (n = 3).
10
Immunoglobulin and FcγRIII Detection
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To determine the presence of the IgG subclasses, as well as the presence of the FcγRIII in the nasal cavity of the BALB/c mice, 5-µm head slides of the different groups were obtained, following the procedures reported by (Carrasco-Yepez et al. 2019 (link)). Slides were blocked with 2% BSA for 1 h; samples were washed with PBS-T and subsequently incubated with rabbit anti-FcγRIII antibodies (1:500) (Abcam)/polyclonal hamster anti-N. fowleri (1: 250)/rabbit anti-mouse IgGt (1:1000)/rabbit anti-mouse IgG1 (1:500)/IgG2a (1:250)/IgG3 (1:500) (Thermo Fisher). Subsequently, the slides were incubated with secondary antibodies coupled to fluorochromes (Alexa 488® (green)/Alexa 647® (red)) (1:1000) and mounted with 4′,6-diamidino-2-phenylindole (DAPI) and VECTASHIELD (Vector Labs). Images were visualized using the Axioskop 2 mot plus confocal fluorescence microscope (Carl Zeiss, Mexico City).
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