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Axio examiner z1 microscope

Manufactured by Zeiss
Sourced in Germany

The Zeiss Axio Examiner Z1 is a high-performance upright microscope designed for advanced research applications. It features a stable, modular design that supports a range of accessories and imaging techniques, including brightfield, darkfield, and fluorescence microscopy. The microscope's key specifications include a high-resolution, high-contrast optical system, a motorized focusing mechanism, and compatibility with a variety of sample types and preparation methods.

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15 protocols using axio examiner z1 microscope

1

Immunofluorescence Analysis of Muscle Cryosections

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For immunofluorescence analysis, all muscles were quick-frozen in prechilled isopentane and embedded in Tissue-Tec (Leica Instruments, Wetzlar, Germany). Muscles were cryotome-sectioned to 10 μm slices. Unfixed sections were rinsed with PBS and permeabilized for 5–10 min in PBS supplemented with 0.1% Triton X-100, then blocked in 10% fetal calf serum (FCS) and 1% bovine serum albumin (BSA) for 1–2 h. Cryotome sections were used for immunofluorescence stainings. For immunofluorescent stainings, sections were washed for 5 min in PBS, permeabilized in 0.1% Triton X-100 for 10 min, and blocked in PBS solution containing 10% FCS and 1% BSA for 1 h at 25 °C. Rhodamine- or Alexa 647 conjugated bungarotoxin 1:2.500 (Rh BTX; Thermo Fisher Scientific, Schwerte, Germany) was used for NMJ detection. Stained cryosections were analyzed and documented using a Zeiss Axio Examiner Z1 microscope (Carl Zeiss MicroImaging)) equipped with an AxioCam MRm camera (Carl Zeiss MicroImaging) and ZEISS AxioVision Release 4.8 (Carl Zeiss MicroImaging). Nuclei diameters were analyzed with the Scion Image Software (Scion Corporation, Frederick, MD, USA).
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2

Muscle Histochemical Staining Protocol

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Modified Gomori trichrome staining was perfomed by incubation of muscle sections in Shandon hematoxylin (Fisher Scientific GmbH, Schwerte, Germany) for 15 min, followed by a 15 min wash in tap water and 45 min incubation in Gomori solution (Sigma-Aldrich Chemie GmbH), washed in tap water, and incubated in 100% ethanol.
For nicotinamide adenine dinucleotide (NADH) dehydrogenase staining, sections were incubated for 30 min at 37 °C in a solution containing NBT (Sigma-Aldrich Chemie GmbH), 50 mM Tris/HCl, and NADH (Sigma-Aldrich Chemie GmbH), then washed in distilled water.
For succinate dehydrogenase (SDH) staining sections were incubated for 45 min at 37 °C in a solution containing Tris 0.2 M pH 7.2, cobalt (II)-chloride, MTT (methylthiazolyldiphenyl-tetrazolium bromide), and NADH. Afterwards, sections were incubated for 30 min in 4% PFA and washed in H2O.
Stained cryosections were analyzed and documented using a Zeiss Axio Examiner Z1 microscope equipped with an AxioCam MRm camera and ZEISS AxioVision Release 4.8 (Carl Zeiss MicroImaging, Göttingen, Germany). Densitometric quantifications were done using ImageJ [20 (link)].
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3

Confocal Imaging Protocols and Techniques

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All confocal images were acquired using a Leica SP8 confocal microscope and LAS X software, with a 25x FLUOTAR water immersion lens (Leica 15506374). Full images of scales were acquired at 0.75x zoom, resulting in 0.606 μm pixel size. Videos of radii were captured at 3x zoom, resulting in 0.152 μm pixel size. Whenever possible, a z step size equivalent to pixel length and width was used to ensure cubic pixels and allow three-dimensional image permutation and rotation. When time constraints required, z step sizes up to the optical section size (1.7 μm at 1 AU pinhole width) were used. A Zeiss AxioZoom V16 and Zen Pro 2012 software was used to acquire certain low-magnification images. Zeiss Axio Examiner Z1 microscope was used for Kaede photoconversion (described above in Zebrafish section).
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4

Confocal Imaging Using Zeiss Axio Examiner

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A fixed-stage Zeiss Axio Examiner Z.1 microscope (Carl Zeiss Microscopy GmbH, Germany) equipped with a confocal scanning unit (Yokogawa CSU-X1; Yokogawa Electric Corporation, Japan) was used for all microscopic analyses. Confocal images were captured using a high-speed digital camera (CCD C9300-221; Hamamatsu Photonics K.K., Hamamatsu City, Japan).
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5

Diaphragm-Phrenic Nerve Preparation and Tubocurarine Effects

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Diaphragm-phrenic nerve preparations were maintained ex vivo in Liley’s solution gassed with 95% O2, 5% CO2 at room temperature [21 (link)]. The recording chamber had a volume of approximately 1 mL and was perfused at a rate of 1 mL/min. The nerve was drawn up into a suction electrode for stimulation with pulses of 0.1 ms duration. The preparation was placed on the stage of a Zeiss Axio Examiner Z1 microscope (Carl Zeiss MicroImaging, Göttingen, Germany) fitted with incident light fluorescence illumination with filters for 547 nm/red (Zeiss filter set 20) fluorescing fluorophore (Carl Zeiss MicroImaging). At the beginning of the experiment, the compound muscle action potential (CMAP) was recorded using a micropipette with a tip diameter of approximately 10 µm filled with bathing solution. The electrode was positioned so that the latency of the major negative peak was minimized. The electrode was then positioned 100 µm above the surface of the muscle, and CMAP was recorded.
For recordings in the presence of tubocurarine, the chamber was filled with 2 mL (300 nM, 500 nM, 800 nM, or 1000 nM) of d-tubocurarine chloride (Sigma Aldrich Chemie, München, Germany). During the curare treatment, trains of 25 repetitive nerve stimulations (5 Hz) were performed at 2 min intervals, and the ratio of CMAP amplitudes (mean (20th–25th)/2nd) was calculated [22 (link)].
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6

Phrenic Nerve-Diaphragm Muscle Preparation

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Diaphragm-phrenic nerve preparations were maintained ex vivo in Liley's solution gassed with 95% O2, 5% CO2 at room temperature (55 (link)). The recording chamber had a volume of approximately 1 ml and was perfused at a rate of 1 ml/min. The nerve was drawn up into a suction electrode for stimulation with pulses of 0.1 ms duration. The preparation was placed on the stage of a Zeiss Axio Examiner Z1 microscope (Carl Zeiss MicroImaging) fitted with incident light fluorescence illumination with filters for 547 nm/red (Zeiss filter set 20) fluorescing fluorophore (Carl Zeiss MicroImaging). At the beginning of the experiment, the compound muscle action potential (CMAP) was recorded using a micropipette with a tip diameter of approximately 10 μm filled with bathing solution. The electrode was positioned so that the latency of the major negative peak was minimized. The electrode was then positioned 100 μm above the surface of the muscle, and CMAP was recorded. For recordings in the presence of d-tubocurarine, the chamber was filled with 2 ml (300, 800 or 1000 nM) of d-tubocurarine chloride (Sigma Aldrich). During the curare treatment, trains of 25 repetitive nerve stimulations (5 Hz) were performed at 2 min intervals, and the ratio of CMAP amplitudes (mean (20th–25th)/2nd) was calculated (44 (link),56 (link)).
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7

Vimentin Surface Visualization Assay

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To visualize surface vimentin, cells were fixed at approximately 50–70% confluency in 4% paraformaldehyde for 30 min at 37°C. Following fixation, to avoid non-specific binding cells were incubated with blocking-buffer (1% BSA in PBS) for 30 min at RT. After blocking, cells were incubated overnight at 4°C with primary rabbit monoclonal and polyclonal anti-vimentin antibodies at 1:500. Next, cells were incubated for 1h at RT with an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at 1:1000. Counterstain was performed with DAPI nuclear stain. To differentiate surface vimentin from intracellular vimentin, additional staining was performed with addition of permeabilization step with 0.1% Triton X-100 in PBS for 15 minutes. Images were captured with a fixed-stage Zeiss Axio Examiner Z.1 microscope (Carl Zeiss Microscopy GmbH, Germany) and confocal scanning unit (Yokogawa CSU-X1, Yokogawa Electric Corporation, Japan).
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8

Visualizing Surface and Intracellular Vimentin

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To visualize surface vimentin, cells were first exposed to a primary anti-vimentin antibody for 1 hours. Primary antibodies for immunostaining included the Novus Biological chicken antibody or Pritumumab. Cells were then fixed in 4% paraformaldehyde for 30 min at 37°C and stained with the secondary antibody, either anti-chicken Alexa Fluor 488 or anti-human Alexa Fluor 488, as appropriate. Counterstain was performed with DAPI nuclear stain. In some cases, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes after fixation and stained with either phalloidin to label F-actin or a second anti-vimentin antibody to label intracellular vimentin. Images were captured with a fixed-stage Zeiss Axio Examiner Z.1 microscope (Carl Zeiss Microscopy GmbH, Germany) and aconfocal scanning unit (Yokogawa CSU-X1, Yokogawa Electric Corporation, Japan). Super resolution images were captured using an Zeiss LSM 980 (Carl Zeiss, Germany) confocal microscope with Airyscan 2. The Zeiss 980 is equipped with a T-PMT, MA-PMTs, a GaAsP detector, and Airyscan 2 with multiplex modes 4Y and 8Y. Optics used are a Pl APO 63x/1.4 NA oil DIC. Zeiss Zen Blue 3.2 and 3.3 were used to capture the images.
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9

Multiphoton Imaging of Spheroid Invasion

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Spheroids of H1299 shLKB1 and pLKO.1 or H157 stable cells were dyed using 1 μM of Red CellTracker (Invitrogen). The H1299 stable spheroids were imaged at 0, 6, and 21 hours post-invasion, and H157 stable spheroids were imaged at 0 and 24 hours post-invasion, using a standard upright Zeiss Axio Examiner Z1 microscope with 20x water immersion objective (1.0 NA DIC (UV) VIS-IR). The second harmonic generation (SHG) signal was obtained using a bandpass 380–430 nm cube. To image the cells stained with Red CellTracker, a bandpass of 570–610 nm cube with a long pass of 550 nm was used. Images were taken with a Coherent Chameleon Verdi laser at 790 nm wavelength. Each multiphoton microscopy image is 512 × 512 pixels in size (425 × 425 μm); each pixel is 0.83 μm. Z-stack images were taken with a 1 μm interval.
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10

Second Harmonic Generation Imaging of H&E Sections

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Second harmonic generation (SHG) images of H&E sections were taken using a Zeiss Axio Examiner Z1 microscope with 20× water immersion objective (1.0 NA DIC [UV] VIS-IR) as described (33 (link)). The SHG signal was obtained using a band pass cube of 380–430nm. Images were taken with a Coherent Chameleon Verdi laser at 820 nm wavelength. Z-stack images were taken with a 1μm interval.
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