Cytosolic [Ca2+] changes were monitored as ratiometric measurements of Fura-2 fluorescence. Isolated mouse islets were cultured on glass coverslips treated with poly-L -lysine (Sigma-Aldrich) and placed in a thermostatic chamber (Harvard Apparatus, Holliston, MA) before incubation with 2 µM Fura-2/acetoxymethyl ester (AM) for 60 min. After washing, Fura-2 fluorescence of a single islet was imaged with alternate 340/380 nm excitation and 510 nm emission using an Axiovert S100 TV through a 40 × 1.3 NA oil immersion objective (Carl Zeiss GmbH, Jena, Germany) as described [38] (link). After the 22.8 mM glucose stimulation, 1 μM thapsigargin was added to evaluate the contribution of endoplasmic reticulum to cytosolic [Ca2+] changes. Data obtained from islets of at least 3 mice were presented both as 340/380 ratio and normalized to cytosolic [Ca2+] at low glucose.
1.3 na oil immersion objective
Manufactured by Zeiss
Sourced in Germany
The 40 × 1.3 NA oil immersion objective is a high-performance lens designed for use in microscopy. It has a magnification of 40× and a numerical aperture (NA) of 1.3, which allows for high-resolution imaging and a shallow depth of field. This objective is intended to be used with oil immersion techniques to improve image quality and resolution.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Phosphate buffered saline (pbs), by Harvard Bioscience (2 mentions)
Axiovert s100 tv, by Zeiss (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Goat anti mouse alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Dylight 649 conjugated anti horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Axiovert s100tv microscope, by Zeiss (1 mentions)
16 bit ccd camera, by Visitron (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Pascal confocal microscope, by Zeiss (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
C9100 50 emccd camera, by Hamamatsu Photonics (1 mentions)
Ultraview spinning disc confocal unit, by PerkinElmer (1 mentions)
Schneider s insect medium, by Thermo Fisher Scientific (1 mentions)
Spinning disc confocal microscope, by Zeiss (1 mentions)
Gluture topical tissue adhesive, by Zoetis (1 mentions)
10 protocols using 1.3 na oil immersion objective
1
Monitoring Cytosolic Calcium Changes in Islets
2
Larvae Dissection and Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Larvae were reared at 25°C and dissected at the third wandering instar stage. Larvae were dissected in HL3.1 solution (in mM, 70 NaCl, 5 KCl, 10 NaHCO3, 4 MgCl2, 5 trehalose, 115 sucrose, 5 HEPES, pH 7.2) and fixed in 4% paraformaldehyde or as otherwise indicated. Following washes in PBT (PBS containing 0.3% Triton X-100), larvae were blocked for one hour in PBT containing 2% normal goal serum, incubated overnight with primary antibody at 4°C, washed, incubated with secondary antibodies for 2 hr at room temperature, washed, and mounted in Vectashield (Vector Laboratories, Burlingame, CA) for imaging. For Syx4 stainings, Syx4 antibody was preabsorbed on Syx4 null mutant tissue to reduce background staining. Antibodies were as follows: mouse anti-Dlg, 1:1000 (DSHB 4F3; Parnas et al., 2001 (link)); anti-Brp, 1:500 (DSHB nc82; Wagh et al., 2006 (link)); anti-GluRIII, 1:500 (Marrus et al., 2004 (link)); anti-GluRIII-488, 1:500 (Blunk et al., 2014 (link); Marrus et al., 2004 (link)); anti-Syx4, 1:500; rabbit anti-Lva, 1:500 (Sisson et al., 2000 (link)); DyLight 649 conjugated anti-horseradish peroxidase, 1:1000 (Jackson ImmunoResearch, West Grove, PA); Alexa Fluor 488 goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, and Alexa Fluor 546 goat anti-mouse, 1:400 (Thermo Fisher Scientific). Images were acquired with a 40 × 1.3 NA oil-immersion objective (Carl Zeiss, Germany).
3
Measuring Mitochondrial ATP in HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial ATP levels were measured by transfecting HeLa cells with the mitochondrially targeted ATP-sensitive FRET-based probe (Mito-ATeam), as previously described [38 (link)]. Briefly, cells seeded on coverslips were washed in a modified Ringer's medium (in mM: 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, pH 7.3) and images were collected using 440nm excitation and alternate 485/535 nm emission on an Axiovert S100 TV microscope through a ×40, 1.3 NA oil-immersion objective (Carl Zeiss AG, Switzerland) equipped with a 16-bit CCD camera, Xenon lamp and filter-based wavelength switcher (Visitron Systems GmbH, Germany). Minimum FRET emission ratios were obtained by washing cells in Ringer's were glucose was replaced by 2-deoxyglucose and contained 10μg/ml oligomycin. Relative changes in mitochondrial ATP are reported as background-subtracted FRET ratios normalized to baseline and minimum FRET emission ratios where Relative Mito-ATeam FRET = ((F335/485 - Fmin) / Fbaseline). The loss of ATP is reported as Loss ATP = (1 - Relative Mito-ATeam FRET) at t=10min after stimulation. Bars show mean ± SEM of 3 independent experiments.
4
Confocal Imaging of Doxorubicin-Stained Hydrogels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confocal
laser scanning images were acquired on a Zeiss LSM 710 confocal laser
scanning microscope equipped with a Zeiss 40×, 1.3 NA oil immersion
objective, using an excitation wavelength of 488 nm and an emission
filter of 510–570 nm for imaging doxorubicin-stained hydrogels.
Still images were acquired at 536 × 536 pixels (64 μm ×
64 μm). Samples were prepared as described in the general gelation
protocol. Gel samples were mixed by pipetting before deposition into
Ibidi 8-well slides for imaging. Images were taken directly after
mixing and 24 h after the onset of gel formation.
laser scanning images were acquired on a Zeiss LSM 710 confocal laser
scanning microscope equipped with a Zeiss 40×, 1.3 NA oil immersion
objective, using an excitation wavelength of 488 nm and an emission
filter of 510–570 nm for imaging doxorubicin-stained hydrogels.
Still images were acquired at 536 × 536 pixels (64 μm ×
64 μm). Samples were prepared as described in the general gelation
protocol. Gel samples were mixed by pipetting before deposition into
Ibidi 8-well slides for imaging. Images were taken directly after
mixing and 24 h after the onset of gel formation.
5
Quantitative Imaging of Neuromuscular Junctions
6
Visualizing Fluorescent Protein Fusions in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
mCyRFP3 fusions were cloned into pLL3.7 m, a modified form of pLL3.7, using standard molecular biology methods. Fusions were made to human calnexin (NM_001746.3), Lifeact, mouse mannosidase II (NM_008549.2), human laminB1 (NM_005573.2), human pyruvate dehydrogenase (NM_000284), chicken paxillin (NM_204984.1), human histone H2B (NM_021058.3), rat connexin-43 (NM_012326.2), and human α-tubulin (NM_006082). All sequences were gifts of M. Davidson (Florida State University). mCyRFP3 fused to the CAAX membrane-localization signals were subcloned into pcDNA3. HeLa cells were grown and transfected as above, then imaged 24–48 h later in FluoroBrite DMEM with B-27. mCyRFP3 fusions were imaged on an Axio Observer microscope with a 63 × 1.3-NA oil-immersion objective (Zeiss) equipped with an UltraVIEW spinning-disc confocal unit (Perkin-Elmer). Excitation was provided by a 488-nm laser excitation, and emission was collected through a 615/70-nm filter with an C9100-50 EMCCD camera (Hamamatsu). Images were acquired using Improvision Volocity 6.0. Maximal intensity projections of optical sections were generated in the ImageJ program25 (link).
7
Maturation of High-K+ Induced GBs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess whether GBs induced by high-K+ stimulation underwent maturation, we imaged the distribution of pre- and postsynaptic markers in these structures several hours post-stimulation. We tested two solutions to maintain our ex vivo preparations (dissected larvae) during prolonged periods. HL6 solution, more similar to larval haemolymph, initially preserves neuronal excitability but does not maintain the health of the preparation over 5–6 h. As an alternative, we used Schneider’s insect medium (Thermo Fisher), which is known to support long-term culturing of the Drosophila brain77 (link)and larval motor axons78 (link), allowing more than 10 h of viability. Larvae were dissected as previously described using GLUture topical tissue adhesive (Zoetis Inc.) as a replacement for the dissection pins, and then subjected to an incubation period in Schneider’s post-stimulation (30 min, 3, 5 and 10 h). Time-lapse imaging was performed at each timepoint with larvae submerged in Schneider medium, using a spinning disc confocal microscope (Andor) with a 60 × 1.3 NA oil immersion objective (Carl Zeiss). The same NMJs were imaged throughout each timepoint, which were matched retrospectively.
8
Confocal Microscopy Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confocal images were obtained using a laser scanning confocal microscope (LSM 710 mainly; LSM 980 was used for super resolution images) with a 40 × 1.3 NA water-immersion objective or a 63 × 1.3 NA oil-emersion objective (Carl Zeiss). Images were processed in ImageJ/FIJI3 (National Institutes of Health) and Adobe Illustrator (2022 version 26.0.1) and Adobe Photoshop (2020 version 21.2.1) software. The live imaging experiments were performed with a spinning disk confocal microscope (Andor) using a 60 × 1.3 NA oil immersion objective (Carl Zeiss), equipped with a heating stage heated to 25 °C. Quantification of the bouton number was performed at NMJ 6/7, abdominal segments A2–A4 were analyzed. In general, at least 12 (fixed) or 10 (live) NMJs of each genotype were analyzed for each experiment/time-point. The Software packages used were: Black 2011 SP1 for LSM 710, ZEISS ZEN 3.3 (blue edition) for LSM 980 and Andor iQ3 (3.6) for Spinning disk. Quantitative and video analyses were performed using maximum intensity projections from the z-stacks on the image. Images were mounted using Adobe Illustrator and Photoshop.
9
Immunofluorescence Staining of Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were rinsed three times with PBS (Biochrom) and fixed in 4% paraformaldehyde (Carl Roth, Karlsruhe, Germany), permeabilized with 0.1% Triton X100 (Roth, Germany) and blocked 2% BSA (all in PBS, Sigma-Aldrich). Afterwards, cells were stained with DAPI (dilution 1:10,000 in PBS; Invitrogen, Darmstadt, Germany) and Phalloidin conjugated with Alexa Fluor 488 (dilution 1:250 in PBS; Invitrogen) and anti-human alpha-smooth muscle actin (clone 1A4) conjugated with eFluor 660 (dilution 1:200 in PBS; eBioscience, Frankfurt, Germany) for 2 h at room temperature. Afterwards, cells were imaged with 40×/NA 1.3 oil immersion objective (Zeiss, Jena, Germany) using confocal laser scanning microscope LSM700 (Zeiss).
For quantitative analysis of αSMA positive cells, at least 50 cells were manually counted for each independent experiment. At least 3 independent experiments with fibroblasts and CAF from 3 different donors were performed for each condition.
For quantitative analysis of αSMA positive cells, at least 50 cells were manually counted for each independent experiment. At least 3 independent experiments with fibroblasts and CAF from 3 different donors were performed for each condition.
10
Topological Analysis of Cell-free Matrices
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To visualize and analyze topological parameters, cell-free FbColl and HAColl matrices were stained with 50 μM 5-(and 6)carboxytetramethylrhodamine succinimidyl ester (5(6)-TAMRA-SE) (Invitrogen, Carlsbad, USA) at room temperature for 1 h and rinsed 3 times with PBS (Biochrom). Matrices were imaged using a confocal laser scanning microscope (cLSM, LSM700, Zeiss, Jena, Germany) using 40×/NA 1.3 oil immersion objective (Zeiss). Acquired images were 1024 × 1024 pixels in resolution (xyz-voxel size: 0.13 × 0.13 × 0.5 μm) and a vertical stack size of 200 images (equivalent to 100 μm). Pore size and fibril diameter were determined as previously described in ref.
38 and 42 using a home-built image analysis tool using an erosion algorithm and autocorrelation analysis, respectively.
38 and 42 using a home-built image analysis tool using an erosion algorithm and autocorrelation analysis, respectively.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!