All imaging experiments were performed on a Leica TCS SP5 gated-STED microscope, using an HCX PL APO 100×100/1.40/0.70 oil immersion objective lens (Leica Microsystems, Mannheim, Germany). Emission depletion was accomplished with a 592 nm STED laser. Excitation was provided by a white laser at the desired wavelength for each sample. For imaging of MCF10A nuclei, Chromeo488 was excited at 470 nm and its fluorescence emission detected at 480–530 nm, with 1–10 ns time gating using a hybrid detector (Leica Microsystems). Atto532 excitation was performed at 532 nm and the emission collected between 545 and 580 nm by a hybrid detector, with time gating of 2.5–6 ns. The two channels were acquired in line-sequential mode, and the excitation and depletion power were adjusted separately for each channel. 512 × 512 pixel images were acquired with a pixel size of 40 nm. Similar settings were used for imaging the 100- and 20-nm nanorulers.
Hcx pl apo 100 100 1.40 0 70 oil immersion objective lens
Manufactured by Leica
The HCX PL APO 100×100/1.40/0.70 oil immersion objective lens is a high-performance optical component designed for use in microscopy applications. It features a magnification of 100x, a numerical aperture of 1.40, and a working distance of 0.70 mm. This lens is intended for use with oil immersion techniques to achieve high-resolution imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using hcx pl apo 100 100 1.40 0 70 oil immersion objective lens
1
Super-resolution Imaging of Cellular Structures
2
Gated-STED Microscopy Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All imaging experiments were performed on a Leica TCS SP5 gated-STED microscope, using a HCX PL APO ×100 100/1.40/0.70 oil immersion objective lens (Leica Microsystems, Mannheim, Germany). Emission depletion was always accomplished with a 592 nm STED laser.
Excitation was provided by a white laser at the desired wavelength for each sample. Chromeo488 was excited at 488 nm and its fluorescence emission detected at 500–560 nm, with 1–10 ns time gating. Atto532 excitation was performed at 532 nm and the emission collected between 540 and 580 nm, with time gating of 2.5–6 ns. For two-color images, excitation/emission wavelengths were adjusted to 470/480–530 nm and 545/550–585 nm for Chromeo488 and Atto532, respectively.
For M-STED, stacks of STED images at different STED powers were obtained using the line sequential acquisition mode (400–700 Hz). STED power was measured at the aperture of the objective, at the sample plane (Supplementary Fig.16 ). For the images acquired in the presence of STED-induced background, the excitation power was modulated as an exponentially decaying function with time-constant τexc = n/2, where n is the number of images forming the stack.
Excitation was provided by a white laser at the desired wavelength for each sample. Chromeo488 was excited at 488 nm and its fluorescence emission detected at 500–560 nm, with 1–10 ns time gating. Atto532 excitation was performed at 532 nm and the emission collected between 540 and 580 nm, with time gating of 2.5–6 ns. For two-color images, excitation/emission wavelengths were adjusted to 470/480–530 nm and 545/550–585 nm for Chromeo488 and Atto532, respectively.
For M-STED, stacks of STED images at different STED powers were obtained using the line sequential acquisition mode (400–700 Hz). STED power was measured at the aperture of the objective, at the sample plane (Supplementary Fig.
3
Immunofluorescence Staining of PARP-1 in HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% formaldehyde in PBS 1× for 15 minutes and washed several times with PBS 1×. After fixation, HeLa cells were permeabilized and incubated in blocking buffer solution (5% w/v bovine serum albumin, 0.1% (v/v) Triton X‐100 in PBS) for 1 hour at room temperature.
For PARP‐1 detection, cells were incubated overnight at 4°C with the primary antibody mouse anti‐PARP1 (sc‐8007; Santa Cruz Biotechnology), in blocking buffer (1/50 dilution), followed by several washing steps. Cells were then incubated with the secondary antibody Alexa 488‐conjugated anti‐mouse (A28175; Thermo Fisher Scientific) in PBS (1/600 dilution), for 1 hour at room temperature, and washed with PBS.
Cells were stained with TO‐PRO‐3 iodide (T3605; Thermo Fisher Scientific) (dilution 1:2000) in PBS and left incubating for 25 minutes at room temperature and subsequently were washed several times with ultrapure water.
Confocal images of immunostained samples were acquired on a Leica TCS SP5 microscope, using a HCX PL APO × 100 100/1.40/0.70 oil immersion objective lens (Leica Microsystems, Mannheim, Germany). Excitation source was provided by a white laser at the desired wavelength starting from 470 nm. Alexa 488 was excited at 488 nm, and its fluorescence emission detected at 500 to 560 nm. TO‐PRO‐3 iodide excitation was performed at 633 nm, and its emission collected in the band 645 to 710 nm.
For PARP‐1 detection, cells were incubated overnight at 4°C with the primary antibody mouse anti‐PARP1 (sc‐8007; Santa Cruz Biotechnology), in blocking buffer (1/50 dilution), followed by several washing steps. Cells were then incubated with the secondary antibody Alexa 488‐conjugated anti‐mouse (A28175; Thermo Fisher Scientific) in PBS (1/600 dilution), for 1 hour at room temperature, and washed with PBS.
Cells were stained with TO‐PRO‐3 iodide (T3605; Thermo Fisher Scientific) (dilution 1:2000) in PBS and left incubating for 25 minutes at room temperature and subsequently were washed several times with ultrapure water.
Confocal images of immunostained samples were acquired on a Leica TCS SP5 microscope, using a HCX PL APO × 100 100/1.40/0.70 oil immersion objective lens (Leica Microsystems, Mannheim, Germany). Excitation source was provided by a white laser at the desired wavelength starting from 470 nm. Alexa 488 was excited at 488 nm, and its fluorescence emission detected at 500 to 560 nm. TO‐PRO‐3 iodide excitation was performed at 633 nm, and its emission collected in the band 645 to 710 nm.
4
Super-Resolution STED Microscopy Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
STED measurements were performed on a Leica TCS SP5 gated-STED microscope, using an HCX PL APO 100×100/1.40/0.70 oil immersion objective lens (Leica Microsystems, Mannheim, Germany). Emission depletion was performed with a 592 nm CW STED laser. The 592 nm STED laser power was set at 50% of the 350 mW maximum power (∼175 mW). Excitation was provided with a white laser at 488 nm wavelength for Alexa Fluor 488 and with emission detection band 495–525 nm, with 1.50–9.50 ns time gating using a hybrid detector (Leica Microsystems). Typical pixel size is 40 nm, with the pixel dwell time of 2 µs and 48 lines averaging.
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!