After expansion, expanded gels were shrunk with three 5-min baths of 1× PBS. Then, primary antibodies were incubated overnight at 4 °C in PBS with 2% of bovine serum albumin (BSA). Gels were washed three times 5 min in PBS with 0.1% Tween 20 (PBST) prior to secondary antibodies incubation for 3 h at 37 °C. After a second round of washing (three times 5 min in PBST), gels were expanded with three 15-min baths of ddH20 before imaging. Antibodies used are referenced in the Reagents and Tools Table. Image acquisition was performed on an inverted Leica Thunder DMi8 microscope using a 20× (0.40 NA) or 63× (1.4 NA) oil objective with Thunder SVCC (small volume computational clearing) mode at max resolution, adaptive as “Strategy” and water as “Mounting medium” to generate deconvolved images. To avoid gel drifting during acquisition, gels were imaged on Poly-D-lysine (A3890401, Gibco)-coated 24-mm coverslips (0117640, Marienfeld).
Dmi8 thunder microscope
Manufactured by Leica
Sourced in Germany
The Leica DMi8 Thunder is a high-performance microscope designed for advanced imaging and analysis. It features a modular design, allowing for customization to suit various research and laboratory applications. The core function of the DMi8 Thunder is to provide users with superior optical performance, enabling detailed observations and precise measurements of specimens.
Automatically generated - may contain errors
Lab products found in correlation
Las x, by Leica (3 mentions)
Tcs sp8 microscope, by Leica (2 mentions)
Fluorescein isothiocyanate dextran solution, by Merck Group (2 mentions)
Poly d lysine, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Nanozoomer 2.0 ht, by Hamamatsu Photonics (1 mentions)
Thunder imaging system, by Leica (1 mentions)
Slowfade mounting media, by Thermo Fisher Scientific (1 mentions)
D9542, by Merck Group (1 mentions)
Las x software, by Leica (1 mentions)
Dfc9000 gtc scmos camera, by Leica (1 mentions)
Las af, by Leica (1 mentions)
Fluoview 1200, by Olympus (1 mentions)
Nextseq 500, by Illumina (1 mentions)
14 protocols using dmi8 thunder microscope
1
Optimized immunostaining protocol for hydrogel samples
2
High-Resolution Microscopic Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pieces of gels were mounted on 24-mm round precision coverslips (1.5H, 0117640, Marienfeld) coated with poly-d -lysine for imaging. Image acquisition was performed on an inverted Leica TCS SP8 microscope or a Leica Thunder DMi8 microscope using a ×631.4 NA oil objective with Lightening or Thunder LVCC (large volume computational clearing) mode at max resolution, adaptive as ‘Strategy’ and water as ‘Mounting medium’ to generate deconvolved images. Three-dimensional stacks were acquired with 0.12 μm z-intervals and an x, y pixel size of 35 nm (Leica TCS SP8) or 0.21 μm z-intervals and an x, y pixel size of 100 nm (Thunder DMi8).
3
Immunostaining Protocol for Cell Gels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After expansion, human cell gels were shrunk with three 5-min baths of 1× PBS. Then, primary antibodies were incubated for 3 h at 37°C in PBS with 2% of bovine serum albumin (BSA). Gels were washed 3 times 10 min in PBS with 0.1% Tween 20 (PBST) prior to secondary antibodies incubation for 3 h at 37°C. After a second round of washing (3 times 10 min in PBST), gels were expanded with three 30-min baths of ddH20 before imaging. Antibodies used are referenced in S2 Table . Tubulin staining presented in the figures corresponds to either a mixture of anti α- and β-tubulin (raised in mouse) or anti α-tubulin alone (raised in rabbit), depending on the species of the other protein stained.
For retina slices immunostaining, primary antibody incubation was prolonged overnight at 4°C. Image acquisition was performed on an inverted confocal Leica TCS SP8 microscope or on a Leica Thunder DMi8 microscope using a 20× (0.40 NA) or 63× (1.4 NA) oil objective with Lightning or Thunder SVCC (small volume computational clearing) mode at max resolution, adaptive as “Strategy” and water as “Mounting medium” to generate deconvolved images. 3D stacks were acquired with 0.12 μm z-intervals and an x, y pixel size of 35 nm.
For retina slices immunostaining, primary antibody incubation was prolonged overnight at 4°C. Image acquisition was performed on an inverted confocal Leica TCS SP8 microscope or on a Leica Thunder DMi8 microscope using a 20× (0.40 NA) or 63× (1.4 NA) oil objective with Lightning or Thunder SVCC (small volume computational clearing) mode at max resolution, adaptive as “Strategy” and water as “Mounting medium” to generate deconvolved images. 3D stacks were acquired with 0.12 μm z-intervals and an x, y pixel size of 35 nm.
4
Imaging and Quantifying Cortical and Hippocampal Neuronal Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bright-field images were acquired with an Olympus D72 camera attached to a Nikon Eclipse E600 microscope and with a NanoZoomer 2.0-HT (Hamamatsu Photonics). Fluorescent images were taken using a Leica TCS SP5 confocal microscope, a Nikon Eclipse E600 microscope (BrdU images), a NanoZoomer 2.0-HT, and a Leica Thunder DMi8 microscope attached to a Leica K5, SCMOS camera. Image processing was done with FIJI (ImageJ). To determine the layer distribution of cells in the cortex (Cux1 for upper layers, CTIP2 for lower layers, BrdU, and GFP), images from the primary somatosensory barrel field (SBF1) were taken using 10×, 20×, and 40× oil-immersion objectives on a Leica TCS SP5 confocal microscope. Cortical strips were divided into 10 identical bins of 70 µm width using a macro created in ImageJ (FIJI), and the number of cells in each bin was counted manually. To analyze the distribution of BrdU+ cells in the granule cell layer of the DG (BrdU at E15 and P10–P11) and the CA1 (BrdU at E12 and E15), the cell layers were subdivided into three layers (upper, middle, and inner) and the number of BrdU+ cells in each position was counted. In the DG, the number of BrdU+ cells located in the hilus was also counted. The thickness of the layered hippocampal structures CA1 and DG was determined by measuring the average width of each structure.
5
Silica-Induced Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After stimulation with 50 μg/cm2 silica for 28 days, the polyester (PET) membranes of Transwell inserts were removed for further research. To assess cell proliferation in ALI cultures, we used a 5-ethynyl-2'-deoxyuridine (EdU) detection kit (RiboBio). Fluorescent slides were observed on a Leica Thunder DMi8 microscope. Three duplicates were performed for each group, and twenty randomly selected fields (40× objective) were analyzed to calculate the proportion of positive cells.
6
Advanced 3D Image Reconstruction and Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three-dimensional structures were reconstructed using the microscope software ZEN 3.0 SR black (64 bit) for ZEISS microscopy (Carl Zeiss) and Imaris (Bitplane, Belfast, United Kingdom). For the rendering of three-dimensional images, the maximum projection mode was used for the comparison of treatments (Fig. 1 and Supplementary Fig. 2 ). The dynamic ranges of images were adjusted using the Min/Max function, which can help to automate and optimize the display range for each image. In the other cases, the transparent mode was used to show stereoscopic images, in which a three-dimensional image with a transparent effect was calculated.
Live-cell images taken on the Leica THUNDER DMi8 microscope were processed using the THUNDER Imaging System (Leica Microsystems, Wetzlar, Germany) in SVCC mode.
Live-cell images taken on the Leica THUNDER DMi8 microscope were processed using the THUNDER Imaging System (Leica Microsystems, Wetzlar, Germany) in SVCC mode.
7
Leica Microscopy Image Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Images were taken on a Leica Microsystems THUNDER DMi8 microscope using LAS-X software for processing. Detailed image analysis protocol is provided in the supplemental experimental procedures .
8
Immunofluorescence Staining of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with paraformaldehyde (3% paraformaldehyde and 2% sucrose in 1× PBS) for 20 min, washed twice with 1× PBS, and cells were permeabilized with a permeabilization buffer (1×PBS and 0.2% Triton X-100) for 5 min. Subsequently, cells were washed twice with 1×PBS, and blocked in PBS-T (1× PBS and 0.05% Tween-20) containing 2% BSA and 10% milk for 1 h. Cells were then incubated with the primary antibody diluted in 1× PBS containing 2% BSA and 10% milk at room temperature for 2 h. Coverslips were washed three times with PBS-T before incubation (1 h) with the appropriate secondary antibodies conjugated to fluorophores (Alexa-488 or Cy3). After three washes with PBS-T, cells were stained with DAPI (5 µg/mL, MilliporeSigma #D9542), and the coverslips were mounted using slow-fade mounting media (Thermo Fisher Scientific, # S36936). Images were captured using a Leica DMi8 THUNDER microscope.
9
Quantification of Bacillus subtilis LLO Internalization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
B. subtilis LLO was internalized into J774A.1 cells as described above, using a 96-well black glass-bottom plate (40,000 cells/well; Greiner Bio-One, Austria, cat# 655892). B. subtilis LLO was stained with CellTracker Orange CMRA Dye (CTO, Invitrogen, C34564) as described above then added to J774A.1 cells. Live cell imaging was performed on a Leica DMi8 Thunder microscope equipped with a DFC9000 GTC sCMOS camera and LAS-X software (Leica, Wetzlar, Germany). Cells were maintained at 37 °C and 5% CO2 in Fluorobrite medium during the imaging session. Fluorescent images of CTO were acquired using a TRITC filter set. Brightfield and fluorescent images were acquired consecutively, using a 63x oil objective every 1 h beginning at 1 h post co-incubation and continuing until 4 h post-incubation. Z-stacks were taken at all time points at 0.4 µm steps to confirm B. subtilis LLO presence within cytoplasm. B. subtilis LLO presence in J774A.1 cells was quantified using Fiji (ImageJ) software and cell counter plugin by counting >1.5 µm rods throughout z-depth. An area of 2090 µm by 1254 µm in each well was imaged and used to perform this quantification.
10
Quantitative 3D Microscopy of Hydrogels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GHS was incubated in a fluorescein isothiocyanate–dextran solution (Sigma, MO, USA, with an average molecular weight of 2 MDa) in Ultra‐Pure Milli‐Q water (15 µM). The scaffolds were then imaged using a Leica DMi8 Thunder microscope (Germany). The Leica application suite X (LAS X, version 3.7.4.23463) software was used to construct 3D images from high‐magnification (250×) z‐stacked images (141 z‐slices, increment size = 0.502 µm). The void fraction was then measured as the fraction of occupied void space over the total volume, using LAS X software. Also, for pore detection and reporting equivalent median pore diameter, a MATLAB code was developed and used, wherein 2D fluorescence microscopy images were thresholded, and the pores were detected and masked. The areas of detected pores were obtained and converted to equivalent‐area circles, from which the diameters were calculated. The median of the equivalent diameter distribution was reported.
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!