Cells were generally imaged 48–72 h after transfection with a Leica TSC SP5 inverted confocal microscope, using either a HCX PL APO 63X/numerical aperture 1.40–0.60 or a HCX PL APO ×100/numerical aperture 1.4 oil-immersion objective. Images were acquired by using the Leica AS software. To count ER–mitochondria contacts, a complete z-stack of the cell was acquired every 0.29 µm. Z-stacks were processed using Fiji [64 (link)]: images were first convolved, and then filtered using the Gaussian Blur filter. A 3D reconstruction of the resulting image was obtained using the Volume J plugin (http://bij.isi.uu.nl/vr.htm ). A selected face of the 3D rendering was then thresholded and used to count ER–mitochondria contact sites.
As software
Manufactured by Leica
Sourced in Germany
The AS software is a core component of Leica's lab equipment. It provides essential functions for data analysis and processing. The software's primary purpose is to enable users to effectively manage and interpret data generated from Leica's instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Hcx pl apo 100, by Leica (2 mentions)
Axiovert microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Tsc sp5, by Leica (1 mentions)
Tsc sp5 inverted confocal microscope, by Leica (1 mentions)
Alexa fluor 488 or 594 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Cellmask deep red, by Thermo Fisher Scientific (1 mentions)
8 well chamber, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 protocols using as software
1
Quantifying ER-Mitochondria Contact Sites
2
Confocal Imaging and Quantification of ER-Organelle Contacts
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Cells were imaged with a Leica TSC SP5 inverted confocal microscope, using either a HCX PL APO 100×/numerical aperture 1.40–0.60 or a HCX PL APO ×100/numerical aperture 1.4 oil-immersion objective. Images were acquired by using the Leica AS software. To count ER-MT, ER-PM, PO-MT, and PO-ER contacts, a complete Z-stack of the cell was acquired every 0.29 μm and processed using ImageJ National Institutes of Health (NIH). Images were first convolved, and the cells were selected using the freehand selection of ImageJ in the drawing/selection polygon tool and then processed using the “Quantification 1” plugin (https://github.com/titocali1/Quantification-Plugins accessed on 10 December 2021). A 3D reconstruction of the resulting image was obtained using the Volume J plugin (https://github.com/titocali1/Quantification-Plugins accessed on 10 December 2021). A selected face of the 3D rendering was then thresholded and used to count short and long contact sites through the “Quantification 2” plugin (https://github.com/titocali1/Quantification-Plugins accessed on 10 December 2021).
3
Immunofluorescence Staining of Transfected HeLa Cells
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Transfected or TAT α-syn loaded HeLa cells plated on coverslips were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS; 140 mM NaCl, 2 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, pH 7.4) for 20 min and washed three times with PBS. Cell permeabilization was performed by 20 min of incubation in 0.1% Triton X-100 PBS followed by 30 min wash in 1% gelatin (type IV, from bovine skin, Merck KGaA, Darmstadt, Germany) in PBS at room temperature. The coverslips were then incubated for 90 min at 37 °C in a wet chamber with the specific antibody diluted in PBS. Staining was revealed by the incubation with specific AlexaFluor 488 or 594 secondary antibodies for 45 min at room temperature (1:100 dilution in PBS; Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence was analyzed with a Zeiss Axiovert microscope equipped with a 12-bit digital cooled camera (Micromax-1300Y; Princeton Instruments Inc., Trenton, NJ, USA) or Leica Confocal SP5 microscope. Images were acquired by using Axiovision 3.1 or Leica AS software (Leica Microsystems, Wetzlar, Germany).
4
Qualitative Nanoparticle Uptake in Tenocytes
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Qualitative uptake of NPs was studied by confocal
imaging in human tenocytes. Cells (passage #5) were seeded at a concentration
of 3 × 105 in an 8-well chamber (Thermo Fisher Scientific,
USA) and allowed to attach overnight. 200 μL of samples (25
μg/mL of NPs and DMEM-F12 as a negative control) was added,
and cells were incubated at 37 °C for 1 h. Then, cells were washed
with PBS, and the cell membrane was stained with 200 μL CellMask
Deep Red (50 ng/mL, Thermo Fisher, USA) for 3 min at 37 °C. Samples
were washed with PBS, and cells were fixed with 200 μL of a
solution of 4% (v/v) of PFA (Sigma-Aldrich, USA) for 15 min at 37
°C. After washing, the nucleus was stained with DAPI (Thermo
Fisher Scientific, USA) at concentration of 2.5 μg/mL for 3
min, washed twice, and then stored in PBS at +4 °C. The images
were captured with a Leica TCS SP8 STED 3X CW 3D Inverted Microscope
(Leica Microsystems, Germany), using a 63× water objective, and
analyzed with Leica AS software (Leica Microsystems, Germany).
imaging in human tenocytes. Cells (passage #5) were seeded at a concentration
of 3 × 105 in an 8-well chamber (Thermo Fisher Scientific,
USA) and allowed to attach overnight. 200 μL of samples (25
μg/mL of NPs and DMEM-F12 as a negative control) was added,
and cells were incubated at 37 °C for 1 h. Then, cells were washed
with PBS, and the cell membrane was stained with 200 μL CellMask
Deep Red (50 ng/mL, Thermo Fisher, USA) for 3 min at 37 °C. Samples
were washed with PBS, and cells were fixed with 200 μL of a
solution of 4% (v/v) of PFA (Sigma-Aldrich, USA) for 15 min at 37
°C. After washing, the nucleus was stained with DAPI (Thermo
Fisher Scientific, USA) at concentration of 2.5 μg/mL for 3
min, washed twice, and then stored in PBS at +4 °C. The images
were captured with a Leica TCS SP8 STED 3X CW 3D Inverted Microscope
(Leica Microsystems, Germany), using a 63× water objective, and
analyzed with Leica AS software (Leica Microsystems, Germany).
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