Lasaf 2

Manufactured by Leica

Sourced in Germany

LASAF 2.7.3 is a Leica software program designed for advanced image acquisition, processing, and analysis. It provides a user-friendly interface and a range of tools for managing and manipulating digital images obtained from Leica microscopy systems.

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40 protocols using lasaf 2

Confocal Imaging of Retinal and Cortical Tissue

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Stained and flat mounted retinas were imaged at high resolution with a Leica TCS SP5 confocal microscope equipped with the following objective lenses: ×10 HC PL APO Numerical Aperture (NA) 0.4, ×20 HCX PL S-APO NA 0.5, ×40 HCX PL APO NA 1.25, and ×63 HCX PL APO NA 1.40.
The cortex from stained brain vibratome sections was imaged with a Zeiss LSM880 confocal laser scanning microscope equipped with the following objective lenses: ×10 Plan Apo NA 0.45, ×20 Plan Apo NA 0.80, ×40 C Apo NA 1.20, and ×63 Plan Apo NA 1.40.
Low magnification, whole-retina pictures were acquired with a MZ16F stereomicroscope (Leica) coupled to a digital camera (Hamamatsu, C4742-95).
Image acquisition, analysis and processing was performed using LAS-AF 2.6 (Leica), ZEN Software 2.3 SP1 (Carl Zeiss), Volocity 6.3 (Perkin Elmer), Photoshop CS6 13.0 (Adobe), Illustrator CS6 16.0.0 (Adobe) and Fiji-IJ 2.0.0-rc-43^{70 (link)} software. All confocal images shown where immunostaining levels are compared or quantified are representative of 6–12 different images from three or more replicate experiments (animals) per group. Settings for scanner confocal detection and laser excitation were always kept identical between samples whenever comparisons between mutant mice and their respective controls were done.

Eilken H.M., Diéguez-Hurtado R., Schmidt I., Nakayama M., Jeong H.W., Arf H., Adams S., Ferrara N, & Adams R.H. (2017). Pericytes regulate VEGF-induced endothelial sprouting through VEGFR1. Nature Communications, 8, 1574.

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Immunofluorescence Assay for Nestin and Pou5f1

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For Nestin and Pou5f1 ( also known as Oct3-4) immunofluorescence, the cells were fixed in 4% PFA and permeabilized with 0.1% Triton X-100/PBS. Cells were, then, blocked with 10% normal goat serum (NGS)/ 1% BSA(bovine serum albumin)/ PBS. Later, the cells were incubated with the primary antibodies (i.e., anti-Nestin, anti-Pou5f1, conjugated respectively with Alexa Fluor 594, sc-33677 AF594 and Alexa Fluor 488, sc-5279 AF488). After 3 washes with 0.1% Triton X-100/PBS cells were incubated with DAPI for 5 min. Images were captured with a fluorescence microscope (DMI6000B; Leica) run with the LAS AF 2.6 software (Leica).

Acurzio B., Verma A., Polito A., Giaccari C., Cecere F., Fioriniello S., Della Ragione F., Fico A., Cerrato F., Angelini C., Feil R, & Riccio A. (2021). Zfp57 inactivation illustrates the role of ICR methylation in imprinted gene expression during neural differentiation of mouse ESCs. Scientific Reports, 11, 13802.

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Non-invasive 3D Imaging of Pine Cone Structure

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Each layer of the inner structure of the hemisected pine cones was observed without any chemical treatment using confocal laser scanning microscopy (Leica Microsystems Ltd. TCS SP5 II MP with SMD, Germany) with a HC PL FLUOTAR 10 × DRY objective lens (Leica Microsystems Ltd. HC PL FLUOTAR 10 × 0.3 DRY, Germany). The Each layer was magnified using a 20 × zoomed lens. Field of view (FOV) was 775 μm × 775 μm × 408 μm; the structures were consecutively captured with 3 μm depth interval. The laser power was 3.4 kW (780 nm), and total exposure time was 185 seconds.
The acquired images were analyzed and processed using the LAS AF 2.7 software (Leica Microsystems Ltd. Germany). To improve the image quality, outlier noise was removed by filter of Image J software (National Institutes of Health, USA). The images that consecutively captured with 3 μm depth interval were reconstructed a 3D morphological structure and they were handled to make movies (Supplementary Videos 1 and 2) with Image J and Photoscape (MOOII tech, Korea).

Song K., Yeom E., Seo S.J., Kim K., Kim H., Lim J.H, & Joon Lee S. (2015). Journey of water in pine cones. Scientific Reports, 5, 9963.

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3D Imaging of Pine Cone Structures

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Sclerids and fibers of the pine cones were observed using a confocal laser scanning microscopy (Leica Microsystems Ltd. TCS SP5 II MP with SMD, Germany) with a HC PL FLUOTAR 10× objective lens (Leica Microsystems Ltd. HC PL FLUOTAR 10×0.3 DRY, Germany) without any chemical treatment. The samples were magnified using a 20× zoomed lens and the corresponding FOV was 775 μm × 775 μm × 408 μm. By using the 3.4 kW (780 nm) laser power, the structures were consecutively captured with 3 μm depth interval and the total exposure time was 185 seconds.
The acquired images were analyzed and processed using the LAS AF 2.7 software (Leica Microsystems Ltd. Germany). Outlier noises were removed by using filters of Image J software (National Institutes of Health, USA) to improve the image quality. The consecutively captured images were reconstructed into a 3D morphological structure.

Song K., Chang S.S., Roper M., Kim H, & Lee S.J. (2017). A Biologically-Inspired Symmetric Bidirectional Switch. PLoS ONE, 12(1), e0169856.

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Visualizing Sodium Ion Distribution in Root Tissues

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A 3.5 wt.% sodium solution was watered into a pot containing the test samples for 20 weeks to visualize the spatial distribution of Na⁺ ions in the outermost root layer. The root was sectioned transversely by using a microslicer (DTK-1000; Dosaka EM, Kyoto, Japan). The thickness of the sectioned slices was approximately 80 μm. The intracellular Na⁺ specific fluorescent indicator STTMA was used to visualize the localization of intracellular free Na⁺. The sectioned slices were incubated in 20 mM MES (2-[N-Morpholino] ethanesulfonic acid) –KOH (pH 6.5) containing 0.5 M mannitol. The sodium-treated roots were soaked in a fluorescent dye solution at 22 ± 3 °C for 12 h (Fig. S4).
The incubated slices were observed under a two-photon laser scanning microscope (Leica Microsystems Ltd. TCS SP5 II MP with SMD, Germany) with a 20× objective lens. The laser power was 1.9 kW (920 nm), and the total exposure time was 230 seconds. The FOV was 775 μm × 775 μm × 90 μm. Morphological structures were consecutively captured at 1 μm depth intervals. The acquired images were processed using LAS AF 2.7 software (Leica Microsystems Ltd. Germany). Outlier noises were removed by using Image J software (National Institutes of Health, USA) to improve image quality.

Kim K., Seo E., Chang S.K., Park T.J, & Lee S.J. (2016). Novel water filtration of saline water in the outermost layer of mangrove roots. Scientific Reports, 6, 20426.

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Three-Dimensional Cactus Stem Imaging

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A cactus stem was sectioned transversely into several slices by using a microslicer (DTK-1000; Dosaka EM, Kyoto, Japan). The thickness of the sectioned slices was ~80 μm. The sliced samples were observed with an inverted microscope (Zeiss Axiovert 200, Zeiss, Germany) with a ×2.5 (NA = 0.075) objective lens. In addition, 2D sectional images along the depth direction were obtained using a two-photon laser scanning microscope (Leica Microsystems Ltd. TCS SP5 II MP with SMD, Germany) with a ×20 objective lens. The laser power was 1.9 kW (920 nm) and the total exposure time was 230 s. The field of view was 775 × 775 × 90 μm. The morphological structures of the sliced samples were consecutively captured at depth intervals of 1 μm. The acquired images were processed using the LAS AF 2.7 software (Leica Microsystems Ltd., Germany). Outlier noises were removed by using the Image J software (National Institutes of Health, USA) to improve image quality.

Kim K., Kim H., Ho Park S, & Joon Lee S. (2017). Hydraulic Strategy of Cactus Trichome for Absorption and Storage of Water under Arid Environment. Frontiers in Plant Science, 8, 1777.

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Apoptosis Induction in HeLa Cells

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HeLa cells were seeded in 24-well plates (Falcon) 1 day before the experiments. Prior to addition of apoptotic inducers, the cells were stained for 40 min with MG and washed twice with DMEM; 15–30 min prior to the start of the movie, the cells were added to the culture medium: TRAIL, at the appropriate dilution, or 63 ng ml⁻¹ of DRB, or 2.5 µg ml⁻¹ of CHX or a combination of 2.5 µg ml⁻¹ of CHX plus TNF at 20 ng ml⁻¹. HeLa-treated cells were imaged at 15-min intervals for 24 h in a 37 °C humidified chamber in ~5% CO₂. The cells were imaged at 20× magnification (0.4 NA HCX PL FL) on a Leica DMi6000b microscope (Leica MicroSystem) equipped with a Hamamatsu Orca-R2 digital CCD Camera and the images were acquired using the LAS AF 2.7 software (Leica MicroSystem). Time to death was monitored by morphological changes associated with apoptosis. The images were analysed using Fiji 2.0.0-rc-43 software^{47 (link)}. The mitochondrial level was calculated from the first fluorescence image, and the cell fate by morphological changes associated with apoptosis, at the end of the experiment.

Márquez-Jurado S., Díaz-Colunga J., das Neves R.P., Martinez-Lorente A., Almazán F., Guantes R, & Iborra F.J. (2018). Mitochondrial levels determine variability in cell death by modulating apoptotic gene expression. Nature Communications, 9, 389.

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Immunostaining of Sciatic Nerve Sections

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Dissected sciatic nerves were snap-frozen in OCT. A cryostat was used to cut 10 µm thick longitudinal sections which were mounted on slides and post-fixed for 10 minutes in ice cold 4% PFA. Sections were blocked for 1 hour at room temperature in 1x PBS, 20% fetal bovine serum, 2% bovine serum albumin, 1% Triton X-100. Slides were incubated overnight in a humidity chamber at 4°C with primary antibodies (Table 1) and following three PBS washes incubated for 1 hour at room temperature with appropriate fluorophore-conjugated secondary antibodies. Nuclei were identified using DAPI. After PBS washes, slides were mounted in Vectashield (Vector Laboratories). Images were acquired using the ×20 objective on a confocal microscope Leica SP5II running the LAS AF 2.7.9723.3 software (Leica) and analyzed blind to genotypes using the cell counter plugin of the ImageJ2 Fiji software.

Wilson E.R., Nunes G.D., Shen S., Moore S., Gawron J., Maxwell J., Syed U., Hurley E., Lanka M., Qu J., Desaubry L., Wrabetz L., Poitelon Y, & Feltri M.L. (2024). Loss of prohibitin 2 in Schwann cells dysregulates key transcription factors controlling developmental myelination. bioRxiv.

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Immunofluorescence Analysis of Peripheral Nerve Fibers

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Immunofluorescence experiments were performed as described (Della-Flora Nunes et al., 2020) . For teasing, sciatic or tibial nerves were dissected, fixed in 4% PFA for 30 min, washed with PBS and teased in slides coated with (3-Aminopropyl)triethoxysilane (TESPA; Sigma). Coating with TESPA was achieved by subsequently submerging glass slides in acetone for 1 min, 4% TESPA in acetone for 2 min and two times in acetone for 30 sec each. The teasing procedure consisted of placing a small portion of the nerve in a PBS droplet over the TESPA-coated slide, followed by careful mechanical separation of individual fibers, first using insulin syringes (0.3 ml 31 G x 8 mm) and then using modified insulin syringes containing insect pins (Fine science tools #26002-10) attached to their needle. The following primary antibodies were used: rabbit anti-JUN 1:200 (Cell signaling #9165), rabbit anti-p-S6 1:200 (Cell signaling #4858), chicken anti-P0 1:300 (Aves #PZO0308) and mouse ant-MBP 1:300 (Millipore #MAB384). Images from teased fibers were acquired at 40X magnification and 1.5X zoom using a confocal microscope Leica SP5II running the LAS AF 2.7.9723.3 software (Leica). Quantifications were performed using ImageJ Fiji v1.52p (Rueden et al., 2017 , Schindelin et al., 2012) . Four to five fields per animal were analyzed.

Nunes G.D., Wilson E.R., Hurley E., He B., O’Malley B.W., Poitelon Y., Wrabetz L., & Feltri M.L. (2020). mTORC1 and JUN are activated after deletion of Prohibitin 1 in Schwann cells and may link mitochondrial dysfunction to demyelination.

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In vivo Detection of Nitric Oxide

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For NO detection in vivo, Vhcl- and Lov-treated tumor-bearing mice (day 18) were co-injected with FITC-lectin and the NO-sensitive fluorescent probe DAR-1 (320 μg in dimethyl sulfoxide (DMSO); Sigma-Aldrich). After 1 h, mice were perfused by intracardiac injection of PBS, and tumors were excised, fixed with 2% PFA (3 h), and snap-frozen in OCT. Thick sections (40 μm) were analyzed by confocal microscopy (Leica TSC SP5) and quantified with ImageJ; pseudocolor images were obtained with the Leica confocal software (LAS AF 2.6.0).
Quantification of intracellular NO in EC was as described^{65 (link)}. Briefly, 1G11 cells were transduced with Ad-C and Ad-mSOD3 viruses and were incubated the next day (3 h, 37 °C) with the NO-sensitive probe 4,5-diamofluorescein (DAF2, 10 μM; Sigma-Aldrich). Cells were analyzed by FACS, using DAF2-untreated cells as negative control.

Mira E., Carmona-Rodríguez L., Pérez-Villamil B., Casas J., Fernández-Aceñero M.J., Martínez-Rey D., Martín-González P., Heras-Murillo I., Paz-Cabezas M., Tardáguila M., Oury T.D., Martín-Puig S., Lacalle R.A., Fabriás G., Díaz-Rubio E, & Mañes S. (2018). SOD3 improves the tumor response to chemotherapy by stabilizing endothelial HIF-2α. Nature Communications, 9, 575.

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