Confocal immunofluorescence images were taken with a Leica TCS SP8 X confocal microscope (Leica; Wetzlar, Germany) using a Leica HC PL APO CS2 63×/1.40 oil immersion objective (Leica; Cat#15506350) and Leica HyD hybrid detectors, or with an Olympus Fluoview 1000 microscope (Olympus; Tokyo, Japan) using a 60×/1.42 oil immersion objective. Laser lines used were AF488 (499 nm), and Cy3 (554 nm). Detection windows used were AF488 (509–544 nm) and Cy3 (564–758) nm. Brightness and contrast were adjusted using Olympus Fluoview software, Leica LAS X software, or ImageJ [15 ,16 (link)].
Hc pl apo cs2 63 1.40 oil immersion objective
Manufactured by Leica
Sourced in Germany
The Leica HC PL APO CS2 63x/1.40 oil immersion objective is a high-performance microscope objective designed for advanced research applications. It features a numerical aperture of 1.40 and a magnification of 63x, providing exceptional optical performance and resolution. The objective is optimized for use with oil immersion techniques to enhance image quality and contrast.
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Lab products found in correlation
Fluoview 1000 microscope, by Olympus (1 mentions)
Las x software, by Leica (1 mentions)
Tcs sp8 x confocal microscope, by Leica (1 mentions)
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
Paraformaldehyde (pfa), by Agilent Technologies (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
A0082, by Agilent Technologies (1 mentions)
Saponin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ds qimc monochromatic camera, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
White light laser, by Leica (1 mentions)
Dapi 4 6 diamidino 2 fenilindol, by Merck Group (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
6 protocols using hc pl apo cs2 63 1.40 oil immersion objective
1
Confocal Microscopy Imaging Protocol
2
Visualizing Intracellular and Extracellular VWF in ECFCs
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ECFCs were plated on rat tail collagen (50 μg/mL) coated glass coverslips. Three different staining procedures were used to detect intra‐ and/or extracellular VWF. Solely intracellular VWF was visualized after fixation and permeabilization with ice‐cold methanol, after which the cells were washed twice with PBS and blocked with PBS, 1% fetal bovine serum (Gibco) and 1% bovine serum albumin (Sigma‐Aldrich). Intra‐ as well as extracellular VWF was visualized after fixation with 4% paraformaldehyde (Alfa Aesar, Karlsruhe, Germany), after which the cells were washed once and blocked and permeabilized with PBS, 5% normal goat serum (DAKO, Glostrup, Denmark) and 0.02% saponin (Sigma‐Aldrich). Extracellular VWF was visualized after fixation with 4% paraformaldehyde, after which the cells were washed once and blocked with PBS and 5% normal goat serum (DAKO). Cells were stained for VWF with polyclonal antibody rabbit anti‐hVWF (A0082, DAKO) and for VE‐cadherin with purified mouse anti‐human CD144 (BD Biosciences) diluted in the corresponding blocking buffer. Nuclear staining was performed with Hoechst (Thermo Fisher Scientific) diluted in PBS. Coverslips were mounted by ProLong® Diamond Antifade Mountant (Thermo Fisher Scientific) and cells were visualized by the Leica TCS SP8 X WLL converted confocal microscope equipped with a HC PL APO CS2 63×/1.40 OIL immersion objective.
3
Confocal Microscopy Imaging Protocol
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Detection of fluorescence signals was performed by an SP8 Tunable Confocal System (TCS: Leica Microsystems, Wetzlar, Germany). The DAPI-stained chromatin was detected between 410 and 470 nm after excitation at 405 nm. The settings for the applied fluorescent dyes conjugated to various secondary antibodies were as follows: Star Green and Alexa Fluor 488—excited at 488 nm, detected from 490 to 560 nm; Star Orange and Alexa Fluor 594—excited at 561 nm, detected from 600 to 660 nm; Star Red and Alexa Fluor 647—excited at 633 nm, detected from 650 to 700 nm. A series of confocal images (‘z-stacks’) with a lateral (x and y) resolution of 45 nm and an axial (z) resolution of 200 nm were acquired by an HC PL APO CS2 63×/1.40 oil immersion objective (Leica). The size of the confocal aperture was set to 1.35 Airy Units (128.9 µm). Image acquisition was carried out by bidirectional scanning along the x-axis and the images were averaged from three distinct frames to reduce image noise. Z-stacks were subjected to deconvolution by Huygens Essential v18.04 (Scientific Volume Imaging, Hilversum, the Netherlands) and 3D reconstructions were obtained using the Leica Application Suite Advanced Fluorescence software v3.1.5.1638 or the Imaris multidimensional microscopy data analysis software v9.6 (Oxford Instruments, Abingdon, the United Kingdom).
4
Confocal and Widefield Microscopy Protocols
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Confocal microscopy was carried out using a Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany). Series of confocal images (“z stacks”) with a lateral (x and y) resolution of 45 nm and an axial (z) resolution of 200 nm were acquired by a HC PL APO CS2 63×/1.40 oil immersion objective (Leica Microsystems GmbH). Size of confocal aperture was set to 1.35 Airy Unit (128.9 μm). Image acquisition was carried out by bidirectional scanning along the x-axis, and images were averaged from three distinct image frames in order to reduce image noise. Image stack deconvolution was performed using a Huygens Essential software v17.10 (Scientific Volume Imaging, Hilversum, Netherlands). 3D reconstructions were obtained using a Leica Application Suite Advanced Fluorescence software v3.1.5.1638 (Leica Microsystems GmbH).
ImmunoFISH images of wheat–barley introgression lines were captured by widefield microscopy on a Nikon Eclipse 80i epifluorescent microscope equipped with DS-QiMc monochromatic camera (Nikon, Tokyo, Japan). Series of grey-scale images for each colour were captured along the Z-axis, taking 15–20 stacks per colour. Single channel images were pseudocoloured and merged in NIS elements (Nikon). All-in-focus images were created using the extended depth of focus (EDF) module.
ImmunoFISH images of wheat–barley introgression lines were captured by widefield microscopy on a Nikon Eclipse 80i epifluorescent microscope equipped with DS-QiMc monochromatic camera (Nikon, Tokyo, Japan). Series of grey-scale images for each colour were captured along the Z-axis, taking 15–20 stacks per colour. Single channel images were pseudocoloured and merged in NIS elements (Nikon). All-in-focus images were created using the extended depth of focus (EDF) module.
5
Confocal Microscopy for Multicolor Imaging
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The detection of fluorescence signals was performed by an SP8 TCS confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany). The DNA stain DAPI was excited at 405 nm and detected between 410-470 nm. The detection settings for the various labelled secondary antibodies used in the present work were as follows: Alexa Fluor 488 was excited at 488 nm and detected between 490-560 nm; Alexa Fluor 594 was excited at 561 nm, detected between 600-660 nm; abberior STAR RED was excited at 633 nm and detected between 650-700 nm. A series of confocal images (“z stacks”) with a lateral (x and y) resolution of 45 nm and an axial (z) resolution of 200 nm were acquired by an HC PL APO CS2 63×/1.40 oil immersion objective (Leica Microsystems). Image stack deconvolution was performed using the Huygens Essential software v18.04 (Scientific Volume Imaging, Hilversum, the Netherlands). No further manipulation was performed on the images.
6
Immunofluorescent Imaging of Mitochondrial Sirt3
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Immunofluorescence analysis was performed as described previously (17) . Briefly, cells were labelled with 200 nM MitoTracker Deep Red (ThermoFisher Scientific, USA), fixed with 2% paraformaldehyde, incubated with anti-Sirt3 primary polyclonal antibody (dilution 1:100, Santa Cruz, USA), followed by FITC-labeled Goat anti-Mouse IgG secondary antibody (dilution 1:100, Proteintech, USA). DAPI (4,6-diamidino-2-fenilindol, Sigma Aldrich, St. Louis, MO, USA) was used for nuclear staining. Confocal imaging was performed by sequential scanning using Leica TCS SP8 X laser scanning microscope, equipped with a HC PL APO CS2 63×/1.40 oil immersion objective and a white light laser (Leica Microsystems, Germany). The excitation wavelengths and emission detection ranges used were 405 nm and 420-477 nm for DAPI, 490 nm and 500-600 nm for FITC, 644 nm and 665-780 nm for MitoTracker Deep Red.
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