MC3T3-E1 S4 cells (passage 35) were seeded at 15% confluence onto UV-sterilized control and nanofiber coverslips. Twenty-four hours after seeding, the cells were fixed and immunostained. Protease and phosphatase inhibitors were used in the initial washing steps to prevent deactivation of Rac1. The following antibodies were used to determine localization of active and total Rac1: New East Biosciences anti-active Rac1-GTP mouse monoclonal antibody (dilution 1:1,000) and Santa Cruz Rac1 rabbit polyclonal antibody (1:200). To ensure cells were adhering to the fibers and not the underlying glass coverslip, the cells were stained with Sigma Aldrich vinculin mouse monoclonal antibody clone hVIN-1 (1:400), DAPI (1:1,000), and phalloidin (1:500). Immunostained slides were then imaged using a 40× water-immersion objective on a Leica DM5500 upright microscope.
Dm5500 upright microscope
Manufactured by Leica
Sourced in Germany
The Leica DM5500 is an upright microscope designed for professional laboratory use. It features a sturdy construction and a range of optical components to provide high-quality imaging and observation capabilities. The DM5500 is suitable for a variety of applications that require precise and detailed microscopic analysis.
Automatically generated - may contain errors
Lab products found in correlation
M165fc microscope, by Leica (1 mentions)
Prism 7, by GraphPad (1 mentions)
Prism seven, by GraphPad (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Tissue plus, by Thermo Fisher Scientific (1 mentions)
Hmscs, by Lonza (1 mentions)
8 protocols using dm5500 upright microscope
1
Monitoring Rac1 Activation on Nanofiber Substrates
2
Quantitative Lipid Staining in C. elegans
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Worms were cultured and maintained at 20°C and fed with E. coli OP50 on NGM plates. Eggs were obtained by alkaline hypochlorite treatment of gravid adults and ∼200 eggs were transferred onto the experimental plates. At L4 larval stage, worms were collected, washed twice with 1 x PBS and then suspended in 120 μL of PBS to which an equal volume of 2X MRWB buffer (160 mM KCl, 40 mM NaCl, 4 mM EGTA, 30 mM PIPES pH 7.4, 1 mM spermidine, 0.4 mM spermine, 2% paraformaldehyde, 0.2% beta-mercaptoethanol) was added for 1 h. After fixation, the worms were freeze-thawed for three times with dry ice and in a 37°C water bath, followed by centrifuging for 1 min at 14,000 x g and then washing once in PBS buffer. Prior to Oil Red O staining, worms were re-suspended and dehydrated in 60% isopropanol for 15 min 250 μL of 60% Oil Red O stain was added to each sample, and samples were incubated overnight at 4°C. Worms were washed twice in 60% isopropanol solution after Oil Red O staining (Li et al., 2018 (link)). Worm images were acquired with the Leica DM5500 Upright Microscope. The lipid staining been quantified with the open-source image analysis software package FIJI/ImageJ from https://imagej.net/Fiji .
3
Zebrafish Melanophore Imaging and Analysis
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Zebrafish adults and embryos were treated with 1 mg/mL epinephrine to contract melanosomes prior to imaging unless otherwise noted. Fish were anesthetized in 0.17% Tricaine in embryo media and positioned in 2.5% methylcellulose in embryo media for imaging. Images of adult fish were captured with a Nikon D90 DSLR camera. Brightfield and incident light images of embryos were captured with Leica M165FC microscope and Leica DFC400 camera. Fluorescent images of embryos were captured with a Leica DM5500 upright microscope with a Leica DFC365FX camera, and a Zeiss Axiovert 200 microscope outfitted with a Yokogawa spinning disk confocal scanner. Images were processed using ImageJ and Leica LAS X software. Cells were counted and analyses were performed using Microsoft Excel and GraphPad Prism 7. Statistical calculations were performed using GraphPad Prism seven as described in each Figure legend.
4
Histological Analysis of Tissue Samples
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Half of the spleen, one lobe of the liver, or part of the tumor was fixed in 4% paraformaldehyde overnight followed by imbedding in paraffin blocks. Paraffin slides were sectioned at a thickness of 4 μm and stained with hematoxylin and eosin (H&E) for pathological analysis at the EPFL Histology Core Facility (HCF). H&E images were captured with the DM5500 Upright Microscope (Leica, Wetzlar, Germany) at the EPFL BioImaging & Optics Platform (BIOP).
5
Immunofluorescence Analysis of Ocular Tissues
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Tissue samples consistent of sclera, limbus and cornea were obtained from the Eye Bank for Sight Restoration and fixed in 4% paraformaldehyde (PFA, FisherScientific) for 24h. The tissue was then dehydrated in 30% sucrose for 24h before cryopreserving in Fisher Healthcare Tissue-Plus O.C.T. Sections of 14 μm were cut and transferred onto glass slides. Sections were re-hydrated by washing 3 × 5 min with PBS before blocking and permeabilizing the cells with 5% normal donkey serum (NDS), 1% BSA and 0.3% Triton X-100 in PBS for 1h at room temperature. Primary antibodies were added diluted in 5% NDS, 1% BSA in PBS and incubated overnight at 4°C. Sections were then washed 3 × 5min with 1% BSA in PBS before adding secondary antibodies and DAPI diluted in 5% NDS, 1% BSA in PBS and incubated for 1h at room temperature in the dark. Lastly the sections were washed 3 × 5 min with 1% BSA in PBS and mounted with ProLong Gold antifade mountant (ThermoFisher). Images were obtained using a Leica DM5500 upright microscope. Tile-scans were taken with 20x and 40x objectives using 10% overlap in both x- and y-directions, before automatically stitching the images in the Leica Micro Systems imaging software. Image processing was performed in FiJi (Schindelin et al., 2012 (link)).
6
Quantifying Protein Micropattern Mechanics
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For tensile testing a custom imaging platform build on a Leica DM5500 upright microscope equipped with a 40X, 0.8 N.A. water immersion objective was used . The mechanical resistance of the different protein micropatterns were either linearly tested (0-10%) or evaluated in cyclically loaded fashion (8% strain, 0.1 Hz). The stretching protocols were executed via a pressure-actuated biaxial stretcher (StageFlexer) at 37°C. To test the potential effect of SDS or trypsin incubation on the pattern integrity, the patterns were treated with 0.1% sodium dodecyl sulfate (SDS) solution (in PBS) or 5% Trypsin (in PBS). The surface strain (deformation beneath and surrounding the pattern) was determined by tracking fluorescent beads on the surface using a custom developed MATLAB algorithm. The normalized mean principal strain drop (MPSD) was defined as the difference between the average surface strain and mean strain beneath the pattern, divided by the average surface strain (Figure 1D). For the protein mat characterization, 15 different patterns per each at least three independent samples were analyzed in each ligand group. For the cell experiments, 32 cells on collagen-pattern and 11 cells on fibronectin-pattern were analyzed from at least three independent experiments.
7
hMSCs Seeding and Immunostaining on Scaffolds
8
Immunofluorescence Staining of Actin and DNA
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A paraformaldehyde fixation buffer (FB) and permeabilization buffer (PB) were prepared for immunofluorescence studies. The FB was made with 3.7% paraformaldehyde in 1× phosphate buffered saline (PBS). The PB buffer was made with 1× PBS, 3% bovine serum albumin, and 0.1% Triton X-100. Excess media was removed with washes of 1× PBS to prevent issues during fluorescence imaging. The samples were fixed for 15 min and blocked with PB for 45 min. Samples were incubated for 30 min in the dark with Phalloidin 565 (1:1000) and DAPI (1:5000) to fluorescently stain actin and double-stranded DNA in the nuclei, respectively. Then the scaffolds were imaged using a Leica DM5500 upright microscope (Leica Microsystems, Buffalo Groove, IL) and analyzed with Leica LASX software.
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