Intravital microscopy was performed using LRP1ctrl and LRP1cKO mice as well as C57Bl/6 mice 2 h after intrascrotal injection of TNFα (500 ng per mouse) as previously described (Weckbach et al., 2014 (link)). For experiments with C57Bl/6 mice, anti-N-MK Ab (Cellmid) or the matching isotype control Ab (clone MOPC-21; 400101; BioLegend) were applied i.p. 12 h before the experiment. Briefly, after cannulation of the carotid artery, the cremaster muscle was surgically prepared as reported previously (Weckbach et al., 2014 (link)). Intravital microscopy was conducted using an upright microscope (Axiotech Vario, Zeiss; and DM6 FS, Leica) at 37°C and recorded using a digital camera (AxioCam HSm, Zeiss; and Zyla sCMOS, Andor Technology). Leukocyte counts were obtained from whole blood (ProCyte Dx; IDEXX Laboratories). Cells attached >30 s were defined as adherent. Diameter of postcapillary venules ranged from 20 to 40 µm. Centerline red blood cell velocities in microvessels were analyzed using fluorescent microspheres (0.5 µm diameter; Polysciences). Blood flow was calculated from the length of at least three microspheres measured in a snapshot image with a defined exposure time and converted offline to mean blood flow velocities. Wall shear rates were calculated as previously described (Weckbach et al., 2014 (link)).
Axiocam hsm
Manufactured by Zeiss
Sourced in Germany
The Axiocam Hsm is a high-speed, high-resolution digital camera designed for microscopy applications. It features a scientific-grade CMOS sensor and can capture images at high frame rates, making it suitable for a variety of live-cell imaging and time-lapse applications.
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Lab products found in correlation
Axio observer z1 inverted microscope, by Zeiss (9 mentions)
Axio observer z1, by Zeiss (6 mentions)
X tremegene hp dna transfection reagent, by Roche (2 mentions)
Axiovision, by Zeiss (2 mentions)
Axiovert 200m cell observer, by Zeiss (2 mentions)
Fluorescent microspheres, by Polysciences (1 mentions)
Procyte dx, by IDEXX (1 mentions)
Clone mopc 21, by BioLegend (1 mentions)
Axiotech vario, by Zeiss (1 mentions)
Dm6 fs, by Leica (1 mentions)
Cal 590 am, by AAT Bioquest (1 mentions)
Axiovert 200m fluorescent, by Zeiss (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Axio observer d1, by Zeiss (1 mentions)
Mzfliii, by Zeiss (1 mentions)
Axiovision ac, by Zeiss (1 mentions)
Calcium green 1 dextran, by Thermo Fisher Scientific (1 mentions)
Axio examiner z1, by Zeiss (1 mentions)
G cepia1er, by Addgene (1 mentions)
24 protocols using axiocam hsm
1
Intravital Microscopy of Leukocyte Dynamics
2
Single-cell cytosolic calcium measurements
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Single-cell cytosolic Ca2+ measurements were performed as previously described38 (link). In brief, SU-DHL-4 cells were loaded with Fura-2 AM as described above. Single-cell imaging was performed using a Zeiss Axio Observer Z1 Inverted Microscope equipped with a 20× air objective and a high-speed digital camera (Axiocam Hsm, Zeiss, Jena, Germany). Data are shown as the ratio of emitted fluorescence of Fura-2 (F340/F380).
3
Calcium Imaging of Murine ESC-Derived Cardiomyocytes
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Murine ESC clones #5, #6 and sham-GFP control differentiated to cardiac tissue were loaded with Cal-590 AM and Ca2+ imaging experiments were performed with only minor modifications to the standard procedures. In brief, cells were seeded on gelatin-coated 12-well plates before differentiation. Following 11 days from cardiac induction, cells were loaded with 1 µM of Cal-590 AM (AAT Bioquest) and 1:2000 of 20% solution in DMSO of Pluronic F-127 (Thermo Fisher Scientific) for 45 min at 37 °C and 5% CO2 in cardiac differentiation medium. After loading the cells de-esterification of the dye was started by replacing the medium with fresh medium lacking phenol red, to minimize interference of with the fluorescent Ca2+ indicator, for 30 min at 37 °C and 5% CO2. The 12-well plates were then mounted on a 37 °C heated stage (Brand and ref) of an inverted microscope (Zeiss Axio Observer Z1) equipped with a 20X air objective and a high-speed digital camera (Axiocam Hsm, Zeiss, Jena, Germany). Cal-590 was excited at the wavelength of 540 nm, and emission was detected at >590 nm. Spontaneous changes in [Ca2+]i are expressed as F/F0, where F is the fluorescence intensity normalized to the resting fluorescence (F0).
4
Intravital Microscopy of Kidney Capillaries
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Intravital video microscopy (IVVM) was performed as described previously26 (link)30 (link). Briefly, following anesthesia with isoflurane, FITC-labeled dextran (2 μmol/kg in 3 ml/kg normal saline) was administered via the penile vein to visualize the capillary vascular space. After 10 minutes, the left kidney was exposed by a flank incision and positioned on a glass stage above an inverted Zeiss Axiovert 200M fluorescent microscope equipped with an Axiocam HSm camera (Zeiss, Germany). Videos of 10 seconds (approximately 30 frames/second) at 200X magnification were acquired from five randomly selected, non-overlapping fields of view. Body temperature was maintained at 35°–37 °C with a warming lamp. Approximately 150 capillaries were randomly selected and analyzed from these fields of view from the kidney of each animal. Capillaries were categorized as “continuous” if red blood cell movement was uninterrupted; “intermittent” if red blood cell movement stopped or reversed; or “no flow” if no red blood cell movement was observed. Data were expressed as the percentage of vessels in each of the three categories.
5
Single-cell ER Ca2+ Measurement Protocol
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Single-cell ER Ca2+ measurements were performed as previously described39 (link). ER Ca2+ levels were measured with the genetically encoded Ca2+ indicator G-CEPIA1er, which was kindly provided by Dr. M. Iino (The University of Tokyo, Tokyo, Japan)26 . The G-CEPIA1er construct was introduced into HeLa cells utilizing X-tremeGENE HP DNA transfection reagent (Roche, Mannheim, Germany, 06366546001) according to the manufacturer’s protocol. A Zeiss Axio Observer Z1 Inverted Microscope equipped with a 20× air objective and a high-speed digital camera (Axiocam Hsm, Zeiss, Jena, Germany) were used for these measurements. Changes in fluorescence were monitored in the GFP channel (480/520 nm excitation/emission). To chelate extracellular Ca2+, EGTA was added to a final concentration of 3 mM. One minute later, 1 µM TG or different SOCE inhibitors were added. All traces were normalized (F/F0) where F0 is the starting fluorescence of each trace.
6
Microvascular Thrombosis in Cremaster Muscle
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Microvascular thrombosis in vivo was investigated in arterioles and venules of the mouse cremaster muscle using a light/dye-injury model as previously described41 , 42 (link). After anesthesia all surgical procedures were conducted on a thermo-controlled plexi-glass stage to maintain body temperature at 37 °C with cover slips for microscopy. Intravital fluorescence microscopy was performed using a modified microscope (Zeiss Axiotech Vario, Carl Zeiss Microscopy GmbH, Germany). Images were recorded with a digital camera (AxioCam HSm, Carl Zeiss Germany) and analyzed with “AxioVision” (Carl Zeiss Microscopy GmbH, Germany). After the surgical preparation of the cremaster muscle 1–2 arterioles and 1–2 venules per cremaster with a diameter of around 50 μm were chosen and FITC-dextran (average molecular weight 150,000 (Sigma-Aldrich, Germany)) in a concentration of 5% in PBS was given in a dose of 1 μl/g mouse via a tail vein catheter. Light with a wave length of 450–490 nm was used to excite the fluorescent dye inducing photochemical injury of the endothelial cell layer. Two end points were defined, first the onset of thrombus formation and second the time until total cessation of blood flow (occlusion time).
7
Contractility and Relaxation of Airway Tissues
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PCLS were removed from the incubator and weighted with steel wires in a 24-well plate to prevent floating and were then transferred to the microplate-holder of an inverted microscope (Axio Observer D1, Carl Zeiss AG, Oberkochen, Germany) with an AxioCam HSm camera (Carl Zeiss AG, Germany). Using the AxioVision software (Version 4.8.2.0, Carl Zeiss AG), airway cross sections were observed for signs of vitality such as beating cilia and spontaneous muscle constrictions. At first, the initial airway area was assessed. Afterwards either the solvent control acetonitrile (1%) or the OP (0.001–100 µmol/L) was added to the slices. After 3 min of incubation time of the OP, ACh (0.5 µmol/L final concentration) was added directly into the culture medium. 2 min after ACh application, a second picture was taken to evaluate time-dependent airway constriction. After 60 min, airway relaxation was calculated using the AxioVision software. Relaxation efficacy of solvent exposed slices was set as 100% and response of all OP-exposed PCLS was related to that value.
8
Fluorescence Microscopy of Cells
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Images were taken using an «Axiovert 200 M Cell Observer» inverted fluorescence microscope with high-speed camera AxioCam HSM of Carl Zeiss company using 60 × /1.4 Oil objective. Diode laser 488 were used for excitation. Images were acquired at a resolution of 1388 × 1040 pixels.
9
Visualizing Zebrafish Caudal Fin Anatomy
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Adult fish were anesthetized and placed on a 1% agarose plate with the caudal fins spread out. Zebrafish embryos, larvae and juveniles were anesthetized in E3 embryo medium containing 0.1 mg/ml tricaine. The plate was placed under a Leica MZ FLIII dissection microscope and images were taken using the AxioCam HSM digital camera and AxioVision AC software (Carl Zeiss). For live confocal imaging, fish were anesthetized and immersed in 0.17mg/ml tricaine in a petri dish. The caudal fins were flattened to the bottom of the petri dish with a slide hold-down (Warner Instruments 64–0248) and imaged with a water-immersion objective equipped on Nikon A1RsiMP Confocal. All images were processed using ImageJ (NIH).
10
Calcium Imaging of Retrogradely Labeled Neurons
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EN cell bodies were retrogradely loaded with Calcium Green-1 dextran (Invitrogen, Eugene, OR, USA) applied as crystals to the peripheral ending of the AVN 24 h prior to an experiment. Imaging of Ca2+ transients was performed with an epifluorescence microscope (Axio Examiner Z1, Carl Zeiss, Germany) and a CCD camera (Axiocam Hsm, Carl Zeiss) in both the absence and presence of locomotor activity. To prevent potential movement artefacts during imaging, all residual muscular elements of preparations were removed. Images were captured at a rate of 10–20 frames s−1 (Axiovision, Zeiss), stored and analysed post hoc using the MBF-ImageJ Java software package (http://rsb.info.nih.gov/ij/ ) and custom-written scripts. The background fluorescence was subtracted, and bleaching effects were corrected using a linear regression algorithm. All data were presented as relative changes in fluorescence (ΔF/F). The duration of an individual Ca2+ transient was taken as the time at half-maximal amplitude of the fluorescence change during a given swimming episode.
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