To determine the time course and efficiency of reversal of zapalog-induced dimerization through photolysis, cells were transfected as above and imaged 24hrs later in a Leica SP8 scanning laser confocal with an HC PL APO 63x/1.40 OIL CS2 objective, set to multichannel imaging of YFP and mCherry with pulsed White Light Laser emission at 514nm and 584nm respectively for a total of 1 frame/sec. 1µM of zapalog was added in dark conditions in order to translocate the YFP from the cytoplasm to the outer membrane of mitochondria, then washed out after 10min by replacing the media with new, pre-warmed media. Using the SP8 FRAP protocol, an ROI of 25µm × 25µm was defined around each cell in the field and a selected cell was then then illuminated separately with a 405nm diode laser (0.5sec @ 30nW), followed by 20 seconds of imaging before repeating the process on the next selected cell.
Hc pl apo 63x 1.40 oil cs2 objective
Manufactured by Leica
Sourced in Germany
The HC PL APO 63x/1.40 OIL CS2 objective is a high-performance microscope objective from Leica. It has a magnification of 63x and a numerical aperture of 1.40, designed for use with oil immersion. The objective is part of Leica's HC (Hybrid Contrast) line and features an APO (Apochromatic) optical design.
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Lab products found in correlation
Tcs sp8 confocal microscope, by Leica (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Microscopy slides, by Thermo Fisher Scientific (2 mentions)
Dapi dye, by Merck Group (2 mentions)
Sp8x confocal microscope, by Leica (2 mentions)
Immu mount, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Las x software, by Leica (2 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (2 mentions)
Ti e inverted microscope, by Nikon (2 mentions)
Las af software, by Leica (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Las 64 bit software package, by Leica (1 mentions)
Professional software version 19, by Scientific Volume Imaging (1 mentions)
Tcs sp8 sted 3 microscope, by Leica (1 mentions)
Application suite x las x version 3, by Leica (1 mentions)
Polyethylenimine (pei), by Thermo Fisher Scientific (1 mentions)
Evos flc microscope, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
20 protocols using hc pl apo 63x 1.40 oil cs2 objective
1
Reversing Zapalog-Induced Dimerization via Photolysis
2
Peroxisomal Membrane Dynamics by FRAP
3
Reversing Zapalog-Induced Dimerization via Photolysis
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To determine the time course and efficiency of reversal of zapalog-induced dimerization through photolysis, cells were transfected as above and imaged 24hrs later in a Leica SP8 scanning laser confocal with an HC PL APO 63x/1.40 OIL CS2 objective, set to multichannel imaging of YFP and mCherry with pulsed White Light Laser emission at 514nm and 584nm respectively for a total of 1 frame/sec. 1µM of zapalog was added in dark conditions in order to translocate the YFP from the cytoplasm to the outer membrane of mitochondria, then washed out after 10min by replacing the media with new, pre-warmed media. Using the SP8 FRAP protocol, an ROI of 25µm × 25µm was defined around each cell in the field and a selected cell was then then illuminated separately with a 405nm diode laser (0.5sec @ 30nW), followed by 20 seconds of imaging before repeating the process on the next selected cell.
4
Peroxisomal Membrane Dynamics by FRAP
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COS7 cells were transfected with a peroxisomal outer membrane marker and zapalog receptor (PEX-mRFP-FKBP) and with cytosolic YFP-DHFR-Myc. 24hrs later, cells were imaged in a Leica SP8 scanning laser confocal with a HC PL APO 63x/1.40 OIL CS2 objective, set to multichannel imaging of YFP and mRFP with pulsed White Light Laser emission at 514nm and 584nm respectively for a total of 1 frame/sec. 1µM of zapalog was added in dark conditions in order to translocate the YFP from the cytoplasm to the outer membrane of peroxisomes (~5min). Then, using the SP8 FRAP protocol, an ROI of 50×50pixels was defined for repeated rounds of 0.5sec illumination with the 405nm diode laser (380nW), followed by 30 seconds of imaging. YFP and mRFP mean intensities in illuminated vs nearby non-illuminated 50×50pxl areas were normalized and average YFP intensity was calculated for each timepoint.
5
Confocal and STED Microscopy Imaging
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For confocal imaging, a Leica TCS SP8 confocal microscope was employed with a Leica HC PL APO 63x/1.40 oil CS2 objective. The images of one technical repetition were acquired at the same resolution format and zoom value for accurate image analysis.
STED imaging was performed with a Leica TCS SP8 STED 3× microscope, equipped with a Leica DFC365 FX digital camera with a STED white CS 100 × 1.40 NA oil objective for optimized overlay of excitation and a STED beam (Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany). Image capturing was performed using the Leica LAS ×64-bit software package. The resolution format of the images is 1024 × 1024 and the corresponding pixel sizes are 18 nm in both x and y. The acquired images were deconvolved using Huygens Professional software version 19.04 (14 September 2021, Scientific Volume Imaging, Hilversum, The Netherlands,http://svi.nl , accessed on 20 January 2023), using the CMLE algorithm, with SNR:07 and 20 iterations.
STED imaging was performed with a Leica TCS SP8 STED 3× microscope, equipped with a Leica DFC365 FX digital camera with a STED white CS 100 × 1.40 NA oil objective for optimized overlay of excitation and a STED beam (Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany). Image capturing was performed using the Leica LAS ×64-bit software package. The resolution format of the images is 1024 × 1024 and the corresponding pixel sizes are 18 nm in both x and y. The acquired images were deconvolved using Huygens Professional software version 19.04 (14 September 2021, Scientific Volume Imaging, Hilversum, The Netherlands,
6
Visualizing NF2 Protein Localization
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24 h before transfection, 5 × 104 HEK293T cells were plated in each well of a 24 well plate (μ-Plate 24 Well Black ID 14 mm, 82426; ibidi). The cells were transiently transfected with N-terminal YFP-fused NF2 WT and mutant proteins using polyethylenimine (PEI, 1 mg/ml, 9002-98-6; Alfa Aesar) at 1:5 ratio of pDNA:transfection reagent. After 7 h, the medium was gently replenished. 24 h post-transfection, the cells were washed with PBS, followed by staining of the nuclei with Hoechst 33342 (20 mM, 12.3 mg/ml, 62249; Thermo Fisher Scientific) at a final concentration of 1 μg/ml according to the manufacturer’s instructions. Fluorescence microscope images were acquired by using a STELLARIS 5 Cryo Confocal Light Microscope (Leica) equipped with a HC PL APO 63x/1,40 OIL CS2 objective. For excitation, a 405 nm (Hoechst 33342) and a 514 nm (EYFP signal) laser were used. The emission range for the channels was set to 420 nm—505 nm (Hoechst 33342), and 545 nm—625 nm (EYFP signal). Image analysis was performed with the Leica Application Suite X (LAS X, version 3.5.5.) software package.
7
Immunofluorescence Staining of Permeabilized Cells
8
Quantification of Neutrophils and Megakaryocytes
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Tissues were fixed in formalin, paraffin-embedded, and sectioned at 5 μm by the University of Toledo Integrated Core Facilities. Neutrophils were observed and enumerated in H&E-stained tissue using an EVOS FLc Microscope (Life Technologies, Carlsbad, CA). For immunofluorescence images, Mks were observed in tongue tissue sections stained overnight at 4 °C with rat anti-CD41 (R&D Systems, Minneapolis, MN) then 1 hour with anti-rat AlexaFluor555 antibody (BioLegend) followed by 4’,6-dia midino-2-phenylindole (DAPI) (Sigma Aldrich) counterstaining. Mounted slides were analyzed on a Stellaris 5 laser scanning confocal equipped with an HC PL APO 63x/1.40 OIL CS2 objective, Power HyD Detectors, and LAS X software (Leica Microsystems, Wetzlar, Germany). Tiling was carried out within LAS X and TauGating was used to separate unwanted background fluorescence.
9
Immunofluorescence Staining of Permeabilized Cells
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Paraformaldehyde-fixed cells were subjected to short cell membrane
permeabilization with 0.5% Triton X-100 in PBS for 5 min. After quenching the
cross-linking with 50 mM glycine, the samples were blocked for 30 min at RT with
5% natural goat serum (NGS) and subsequently incubated with primary antibodies
for 2 hours at RT. After three sequential washing steps with PBS, secondary
antibodies conjugated to a fluorophore were added, together Phalloidin (Abcam)
for staining of actin for 45 min at RT. For nuclear staining, the samples were
incubated with DAPI dye (2 mg/L, Sigma-Aldrich, St. Louis, MO) for 5 min prior
to mounting the coverslips on microscopy slides (ThermoFischer Scientific,
Waltham, MA) with Immu-Mount (Invitrogen, Carlsbad, CA).
Images were obtained using SP8-X confocal microscope (Leica
microsystems, Germany) using a HC PL APO 63x/1.40 Oil CS2 objective. Gain and
offset settings were adjusted according to the fluorescence signal, but they
were kept constant in comparative experimental designs such as
doxycycline-induction tests. Exported files were next subjected to linear
contrast and brightness processing in Photoshop (CS6, 13.0.6 x64, extended) for
image representation purposes.
permeabilization with 0.5% Triton X-100 in PBS for 5 min. After quenching the
cross-linking with 50 mM glycine, the samples were blocked for 30 min at RT with
5% natural goat serum (NGS) and subsequently incubated with primary antibodies
for 2 hours at RT. After three sequential washing steps with PBS, secondary
antibodies conjugated to a fluorophore were added, together Phalloidin (Abcam)
for staining of actin for 45 min at RT. For nuclear staining, the samples were
incubated with DAPI dye (2 mg/L, Sigma-Aldrich, St. Louis, MO) for 5 min prior
to mounting the coverslips on microscopy slides (ThermoFischer Scientific,
Waltham, MA) with Immu-Mount (Invitrogen, Carlsbad, CA).
Images were obtained using SP8-X confocal microscope (Leica
microsystems, Germany) using a HC PL APO 63x/1.40 Oil CS2 objective. Gain and
offset settings were adjusted according to the fluorescence signal, but they
were kept constant in comparative experimental designs such as
doxycycline-induction tests. Exported files were next subjected to linear
contrast and brightness processing in Photoshop (CS6, 13.0.6 x64, extended) for
image representation purposes.
10
Mitochondrial Staining in Cell Cultures
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The cells were seeded on a 4-well chamber slide (Thermo Scientific™ Nunc™ Lab-Tek™ II Chamber Slide and trade, USA) and cultivated for 48 h. With respect to the mitochondrial staining, the cells were incubated with MitoTracker Red CMX Ros (Invitrogen, USA) at 37°C and in a 5% CO2 atmosphere for 30 min. They were then fixed via 4% paraformaldehyde in PBS at RT for 15 min, permeabilised using 0.1% Triton X-100 in PBS (Sigma-Aldrich, USA) at RT for 20 min, and stained using DAPI at 37°C for 15 min (1 : 1 000; Sigma-Aldrich, USA). Confocal images of the cells were acquired using a Leica SP8X microscope (Leica Microsystems, Germany) equipped with a confocal scanning head, a Leica DFC365 FX monochrome digital CCD camera, an HC PL APO CS2 63x/1.40 OIL objective, a 405 nm excitation laser, and a white light laser (WLL; EX 579/EM 599; hybrid detector) (Leica Microsystems, Germany). The images were rendered using the LasX software (Leica Microsystems, Germany).
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