The filamentous actin (F-actin) in the cell cytoskeleton was stained using Alexa Fluor™ 594 Phalloidin (Thermo-Fisher, Waltham, MA, USA, Cat. No. A12381, 200 U/mL) and cell nuclei using DAPI (Thermo-Fisher, D1306, 300 nM concentration). Staining was performed in humidified chambers for 120 min at room temperature. After staining, the samples were rinsed and stored in PBS. Confocal microscopic images were taken using a Nikon CSU-W1 inverted spinning disc confocal microscope based on the Nikon Eclipse Ti2 inverted microscope (Nikon, Tokyo, Japan) with the Yokogawa CSU-W1 spinning disc module (Yokogawa, Tokyo, Japan) and equipped with dual sCMOS PRIME BSI cameras (Teledyne Photometrics, Tucson, AZ, USA). Due to the manner of the samples, Nikon dry objectives CFI Plan Apo VC 20x were used with a 50 mm pinhole disc in Z stack mode (–0–250 mm depth). Images were then processed in Imaris 10.1.0 software (Oxford Instruments, Abingdon-on-Thames, UK).
Cfi plan apo vc 20x
Manufactured by Nikon
Sourced in Japan
The CFI Plan Apo VC 20X is a high-performance objective lens designed for microscopy applications. It features a numerical aperture of 0.75 and a working distance of 1 mm. The lens is constructed with advanced optics to deliver sharp, high-contrast images.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Csu w1 spinning disc module, by Yokogawa (1 mentions)
Imaris 10, by Oxford Instruments (1 mentions)
Dual scmos prime bsi cameras, by Teledyne (1 mentions)
Eclipse ti2 inverted microscope, by Nikon (1 mentions)
Roti plast, by Carl Roth (1 mentions)
Phalloidin ifluor 555, by Abcam (1 mentions)
Spectra x light engine light source, by Lumencor (1 mentions)
Alexa fluor 647, by Abcam (1 mentions)
Anti yap1 antibody, by Proteintech (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Cfi apo tirf 60x oil, by Nikon (1 mentions)
Nile red, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
3 protocols using cfi plan apo vc 20x
1
Cytoskeletal F-actin Visualization in Cells
2
Cross-Sectional Imaging of Date Palm Leaf
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In order to obtain a cross section of a date palm leaflet, it was first precooled (4°C) and fixed with paraffin wax (Roti®-Plast (melting point 56-58°C) from Carl-Roth GmbH + Co. KG, Germany) in a histological sample holder. A 40 μm thick cross section was produced using a microtome (R Jung AG Heidelberg, Germany) and then placed with isotonic 0.9% NaCl (from Carl-Roth GmbH + Co. KG, Germany) water solution on a microscope slide and then covered with a cover glass.
For the acquisition of fluorescence images, a Keyence BZ-8100E fluorescence microscope (Keyence Corp., Osaka, Japan) equipped with a true colours CCD sensor (2/3”, 1.5 megapixels) was used. The following three filters sets (excitation, absorption) were used: DAPI-BP (320-400 nm, 410–510 nm), GFP-BP (430–510 nm, 485–585 nm), Texas-Red (520–600 nm, 570–690 nm) together with a zoom objective CFI Plan Apo VC 20X (Nikon Corp., Tokyo, Japan).
For the acquisition of fluorescence images, a Keyence BZ-8100E fluorescence microscope (Keyence Corp., Osaka, Japan) equipped with a true colours CCD sensor (2/3”, 1.5 megapixels) was used. The following three filters sets (excitation, absorption) were used: DAPI-BP (320-400 nm, 410–510 nm), GFP-BP (430–510 nm, 485–585 nm), Texas-Red (520–600 nm, 570–690 nm) together with a zoom objective CFI Plan Apo VC 20X (Nikon Corp., Tokyo, Japan).
3
Multimodal Immunofluorescence Imaging of Cytoskeleton and Lipid Droplets
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Following culture, cell samples were washed with PBS, fixed in 4% paraformaldehyde, blocked in 2% BSA with 0.5% (v/v) Triton X-100 in PBS for 20 minutes, immersed in 2% BSA for 1 hour, and rinsed in PBS. Staining using the primary anti-TPM1/2 antibody (TM311, Sigma-Aldrich) and anti-YAP1 antibody (Proteintech), and the secondary donkey anti-mouse IgG antibody (Alexa Fluor 647; 1:100; Abcam), donkey anti-rabbit IgG antibody (Alexa Fluor 647; 1:100; Abcam) and donkey anti-mouse IgG antibody (Alexa Fluor 488; 1:100; Abcam) was performed according to manufacturer's protocols. DAPI (Sigma-Aldrich, 20 min) and Phalloidin-iFluor 555 (Abcam) staining (165 nM, 30 minute immersion) was performed in PBS. Lipid droplets staining was performed by immersing cells in 0.1 μg mL -1 Nile red (Sigma) for 5 minutes. Immunofluorescence imaging was performed using a NIKON Ti-E inverted microscope equipped with an sCMOS iXon3 camera (Anodr) and a Spectra X light engine light source (Lumencor). A CFI Apo TIRF 60X Oil (Nikon) and a CFI Plan Apo VC 20X (Nikon) objectives were used. Cell and nucleus projected areas were segmented and quantified using custom-built MATLAB code.
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