A Zeiss 880 laser-scanning confocal microscope (invert configuration), equipped with a 63× oil immersion Plan Apochromat objective lens (numerical aperture = 1.4), was used to acquire high-resolution, 12-bit-depth channel images (image size = 512 × 512). Using the single-track multiple-channel capability of the Zeiss Zen Black software (version 2.3), HA-NK1-eGFP and DyLight 594–labeled ligand were excited simultaneously at 488 and 561 nm, respectively. Well separated spectral emission detection windows (495–555 nm for eGFP and 600–700 nm for DyLight 594) were set up to ensure that the emission signals elicited from each fluorophore were recorded without any bleed through or time delay issues. The pinhole diameter was set to 1 Airy unit, and frame averaging was set to 4 to minimize noise and optimize signal collection. Bright field transmission images were simultaneously detected along with the fluorescence images using the dedicated transmitted light detector.
Plan apochromat objective lens
Manufactured by Zeiss
Sourced in Germany
The Plan-Apochromat objective lens from Zeiss is a high-performance optical lens designed for precise microscopy applications. It is engineered to provide superior image quality with minimal distortion and chromatic aberration across a wide field of view. The lens is optimized for use in advanced microscopy techniques that require accurate and consistent imaging.
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Lab products found in correlation
Petri dish heating stage, by Bruker (2 mentions)
Axiovert 200m microscope system, by Zeiss (2 mentions)
Bioscope catalyst afm system, by Bruker (2 mentions)
Axioscan z1 slide scanning microscope, by Zeiss (1 mentions)
Orca flash 4.0 scmos digital camera, by Hamamatsu Photonics (1 mentions)
Spinning disk confocal microscope, by Zeiss (1 mentions)
Hoechst, by Merck Group (1 mentions)
Acti stain 488 phalloidin, by Cytoskeleton (1 mentions)
Lipidtox deep red, by Thermo Fisher Scientific (1 mentions)
Volocity, by PerkinElmer (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Vectashield mounting medium with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
7 protocols using plan apochromat objective lens
1
Confocal Imaging of NK1 Receptor
2
Quantitative Fluorescence Imaging for Brain Regions
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Fluorescent sections were imaged using an Axioscan.Z1 slide scanning microscope (Zeiss, Thornwood, NY, USA) equipped with an X-Cite XLYS LED light source (Excelitas Technologies, Waltham, MA, USA) and custom filter sets (Semrock). Images were captured using a 20 × /0.8 NA Plan-Apochromat objective lens (Zeiss) and ORCA-Flash4.0 sCMOS digital camera (Hamamatsu) at 325 nm/pixel resolution and were montaged using Zen Blue 2.3 software. Exposure and gain were adjusted using a representative section from a sham-operated animal and were kept constant across all groups. Fluorescence intensity signals for each region of interest (ROI) were quantified for each hemisphere and then averaged. Bisbenzimide staining was used as a guide to trace the ROIs in ImageJ. ROIs were identified using a mouse brain atlas.14 Off-target fluorescence intensity was measured for each section, averaged, and subtracted from the target signal for quantification. The corrected intensity was then averaged across the three sections for each animal and data (mean ± standard error of the mean [SEM]) presented relative to Sham (set to 100%).
3
Quantitative Analysis of Lipid Droplets
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Cryostat sections 20 μm in thickness were rehydrated in PBS for 5 min after which the sections were blocked in blocking buffer (2% BSA, 1% fetal calf serum, 1% goat serum, in 0.5% saponin) for 30 min at RT. The antibody mix (LipidTOX Deep Red (1:400, Life Technologies Europe B.V.); Acti‐stain 488 Phalloidin (1:150, Cytoskeleton Inc.)) was added and incubated for 2 h at RT. After washing with PBS for 5 min, nuclear staining (Hoechst (1:1,000, Sigma‐Aldrich N.V.)) was added for 5 min at RT. Slides were washed in PBS for 5 min, quickly rinsed in water to remove residual salt, and mounted. For each cryosection, Z‐stacks of 5–10 areas were imaged with a spinning disk confocal microscope (Zeiss), using a 40× Plan‐Apochromat objective lens (1.4 Oil DIC (UV) VIS‐IR M27)) at a pixel size of 0.167 μm and at optimal Z‐resolution (240 mm). Z‐stacks were processed in Volocity (PerkinElmer), and the amount of lipid droplets and average size of lipid droplets (depicted as voxels) was calculated.
4
Live Cell Nanomechanical Measurements
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Live cell measurements were performed using a Bruker BioScope Catalyst AFM system (Bruker, Santa Barbara, CA) mounted on an inverted Axiovert 200 M microscope system (Carl Zeiss, Göttingen, Germany) equipped with a Confocal Laser Scanning Microscope 510 META (LSM 510 Meta, Carl Zeiss) and a 40× (0.95 NA, Plan-Apochromat) objective lens (Carl Zeiss). A Petri dish heating stage (Bruker) was used to maintain physiological temperature (37 °C) of cells during measurements. Modified AFM microcantilevers with an attached 25 µm-diameter polystyrene microsphere were obtained from Novascan (Novascan, Ames, IA). The AFM probe spring constant was obtained using the thermal tune method built into the AFM system. Calibrated spring constants for the cantilevers ranged from 0.5 to 1 N/m. After cantilever calibration, the AFM probe was placed on top of the nuclear region of an adherent cell. The deflection setpoint was set between 20 and 25 nm, yielding applied forces between 5 and 18 nN. The force curve ramp rate was set to 0.5 Hz and the probe speed ranged between 1.9 and 2.4 µm/s. Multiple consecutive quasi-static force curves were collected on each individual cell with a deflection trigger of 25 nm.
5
Quantifying Cell Nuclear Mechanics via AFM
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Live cell measurements were performed using a Bruker BioScope Catalyst AFM system (Bruker, Santa Barbara, CA) mounted on an inverted Axiovert 200M microscope system (Carl Zeiss, Göttingen, Germany) equipped with a Confocal Laser Scanning Microscope 510 META (LSM 510 Meta, Carl Zeiss) and a 40x (0.95 NA, Plan-Apochromat) objective lens (Carl Zeiss). A Petri dish heating stage (Bruker) was used to maintain physiological temperature (37 °C) of cells during measurements. Modified AFM microcantilevers with an attached 25 µm-diameter polystyrene microsphere were obtained from Novascan (Novascan, Ames, IA). The AFM probe spring constant was obtained using the thermal tune method built into the AFM system. Calibrated spring constants for the cantilevers ranged from 0.5 N/m to 1 N/m. After cantilever calibration, the AFM probe was placed on top of the nuclear region of an adherent cell. The deflection setpoint was set between 20 nm and 25 nm, yielding applied forces between 5 nN and 18 nN. The force curve ramp rate was set to 0.5 Hz and the probe speed ranged between 1.9 µm/s and 2.4 µm/s. Multiple consecutive quasi-static force curves were collected on each individual cell with a deflection trigger of 25 nm.
6
In Vivo Muscle Oxygenation Measurements
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Measurements of PO2 and VO2 were carried out using an Axioimager-2m microscope with a 20X/0.8 Plan-Apochromat objective lens (Carl Zeiss, Germany). The measurement technique has been described in detail in our previous publications (Golub et al., 2011 (link); Golub and Pittman, 2012 (link); Nugent et al., 2016a (link); Nugent et al., 2016b (link)), except for the application in the current work of the Oxyphor R2 dendrimer phosphorescent probe (www.oxygenent.net ) whose calibration parameters were taken from the manufacturer (Lo et al., 1996 (link)). This R2 probe was chosen for its relatively low molecular weight (2.7 kD) and water-solubility that facilitated its loading into the interstitial space of a thin muscle by topical application of a 10 mg/mL solution for 30 min to the surgically exposed tissue. Octagonal regions of 300 µm diameter, containing no large vessels and separated by about 1 mm from each other were selected for PO2 and VO2 measurement sites in the central region of the muscle. PO2 was sampled at 1 Hz during 300 s of PO2 data collection (Figure 2 ). A color video camera KP-D20B (www.hitachikokusai.com ) was employed for imaging and selection of the measurement sites.
7
Immunofluorescence Staining of CAF-MSCs
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