Mice were euthanized on PND21 with i.p. pentobarbital, lungs were inflated and fixed in 4% paraformaldehyde (PFA) for 24 h. Histomorphometry was carried out on 7-μm paraffin sections of samples (n = 3 per sex/oxygen environment for WT). Samples were stained using hematoxylin and eosin (H&E), cover slipped with Vectamount permanent mounting medium (Vector labs, Newark, CA, USA), and imaged with Leica Thunder Imager DMi8 (Leica, Nußloch, Baden-Wurttemberg, Germany). On serial sections to the sections used for H&E, we performed in situ hybridization (ISH) using the BaseScope Duplex Kit (Advanced Cell Diagnostics, Hayward, CA, USA). Samples (n = 3 per sex/oxygen environment) were treated with a miR-30a probe (Advanced Cell Diagnostics, Hayward, CA, USA) for 2 h at 4 °C. A positive control slide used a mixture of probes targeting housekeeping gene Polr2A. A negative control slide used dapB (a bacterial gene). Nuclei were counterstained with a 50% hematoxylin solution and mounted with Vectamount permanent mounting medium (Vetor labs, Newark, CA, USA). Slides were imaged using Leica Thunder Imager DMi8 (Leica, Nußloch, Baden-Wurttemberg, Germany).
Thunder imager dmi8
Manufactured by Leica
Sourced in Germany
The THUNDER Imager DMi8 is a fully automated inverted fluorescence microscope designed for high-speed, high-resolution imaging of live and fixed cell samples. It features a motorized stage, filter turret, and objective revolver for efficient workflow and data acquisition.
Automatically generated - may contain errors
Lab products found in correlation
Calcein am, by Dojindo Laboratories (2 mentions)
Las x software, by Leica (2 mentions)
Glass coverslip, by Thermo Fisher Scientific (2 mentions)
Levamisole, by Thermo Fisher Scientific (2 mentions)
Gold seal glass microscope slide, by Thermo Fisher Scientific (2 mentions)
Vectamount permanent mounting medium, by Vector Laboratories (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Ab6046, by Abcam (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
K5 camera, by Leica (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Lasx 3, by Leica (1 mentions)
Cls4515, by Corning (1 mentions)
Cm1860 cryostat, by Leica (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Fluoroshield mounting medium with dapi, by Abcam (1 mentions)
Mca970r, by Bio-Rad (1 mentions)
Immersol 518 f immersion oil, by Zeiss (1 mentions)
21 protocols using thunder imager dmi8
1
Lung Histomorphometry and miR-30a Expression
2
Quantifying Spheroid Viability via Live/Dead Fluorescence
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As a gold standard reference measurement of the spheroids viability, live/dead fluorescence assays were performed using the THUNDER imager DMi8 (Leica Microsystems, Wetzlar, Germany) with a microscopic objective lens featuring a numerical aperture (NA) of 0.12. Two contrast agents were applied for three hours before the fluorescence imaging. The first agent is calcein acetoxymethyl (Calcein-AM; Dojindo, Kumamoto, Japan), which stains the living (viable) cells and emits a green fluorescence signal. The second agent is propidium iodide (PI; Dojindo), which contrasts the dead cells by emitting red fluorescence signal.
3
Live/Dead Cell Fluorescence Imaging
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Fluorescence microscopy imaging was performed as a reference gold-standard method for tissue viability visualization. Two fluorescence agents were applied 3 hours before the fluorescence imaging. The first was calcein-acetoxymethyl (calcein-AM; Dojindo, Kumamoto, Japan), which labels living and/or viable cells and emits green fluorescence. The second agent was propidium iodide (PI; Dojindo), which labels dead cells and emits red fluorescence. The fluorescence imaging was performed using a wide-field fluorescence microscope (THUNDER imager DMi8; Leica Microsystems, Wetzlar, Germany) featuring a microscopic objective lens with a numerical aperture of 0.12.
4
Carotid Plaque Immunohistochemical Analysis
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A total of 20 cases of carotid plaques were included which were obtained by randomly selecting 4 cases from each of the 5 groups. This study assessed the extent of macrophage expression by measuring CD68 expression [16 (link)].
Carotid plaques were collected immediately after CEA, fixed in 4% paraformaldehyde, and embedded in paraffin for immunohistochemical staining; specific immunohistochemical steps can be found in theSupplementary Materials .
All tissue sections were observed under a THUNDER imager DMI8 (Leica, Wetzlar, Germany), and all tissue sections were observed at the same magnification (10×) and using the same observation parameters.
Average optical density (AOD) used to represent the protein expression per area within the observed section. The AODs of 4 different proteins (H-FT, L-FT, TfR1, and CD68) were calculated for 20 sections using ImageJ software (1.53q; NIH, Bethesda, MD, USA).
Carotid plaques were collected immediately after CEA, fixed in 4% paraformaldehyde, and embedded in paraffin for immunohistochemical staining; specific immunohistochemical steps can be found in the
All tissue sections were observed under a THUNDER imager DMI8 (Leica, Wetzlar, Germany), and all tissue sections were observed at the same magnification (10×) and using the same observation parameters.
Average optical density (AOD) used to represent the protein expression per area within the observed section. The AODs of 4 different proteins (H-FT, L-FT, TfR1, and CD68) were calculated for 20 sections using ImageJ software (1.53q; NIH, Bethesda, MD, USA).
5
Whole-Mount Immunofluorescence Staining of Oocytes
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For whole-mount immunofluorescence staining, oocytes were cultured in M2 plus or minus 2.5 μM milrinone and collected at the relevant time points. The zona pellucida was removed using the Tyrode’s solution. After a 30-min recovery period, the oocytes were fixed in 2% formaldehyde and 0.1% Triton X-100 in M2 media for 10 min. Cells were washed once in blocking buffer (3–5% BSA and 0.1% Triton X-100 in PBS), before incubation in blocking buffer for 30 min at room temperature (RT). The cells were transferred into the primary antibody solution (3% BSA and 0.1% Triton X-100) and incubated at 4°C overnight. The primary antibody used was a rabbit anti-β-tubulin (Cat. #ab6046; Abcam) at 1:1000. The secondary antibody used was anti-rabbit Alexa Fluor 568 (A11036; Invitrogen) at 1:500. The cells were washed at RT in 0.1% Tween 20 in PBS 3 × 5 min and mounted in Vectashield (Vector Laboratories; H-1000) plus 1 μg/mL DAPI for imaging. Fixed oocytes were imaged with an inverted Leica Thunder Imager DMi8 microscope using a 63 x/1.4 NA oil objective and a sCMOS DFC9000 GT Microscope Camera (Leica) and by a Nikon A1Rsi+ Scanning Confocal Microscope (Nikon Europe B.V.) using a 63 x/1.27 NA oil objective. The brightness and contrast of the images were modified for image preparation.
6
Microglia and Autofluorescence Analysis of Human Brain Tissue
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Human brain tissue for this study was obtained from the Department of Pathology and Laboratory Medicine at UT Health Science Center at Houston. Blocks of tissue were dissected from the brains of three deceased individuals (14-year-old female, 75-year-old male, and 97-year-old female). To be consistent with the animal study, the somatosensory and visual cortex were chosen to perform the microglia and autofluorescence analysis. Formalin-fixed paraffin-embedded sections were immunostained with Iba1 antibody using tyramide signal amplification method (Biotium). Briefly, after deparaffinization, the sections were blocked in 0.3% hydrogen peroxide and blocking buffer (1% bovine serum albumin with 0.5% Triton X-100 in PBS), serially, and then the sections were incubated with anti-human Iba1 mouse monoclonal antibody (1:200; FUJIFILM, catalog no. NCNP27). The primary antibody was detected by horseradish peroxidase–conjugated goat anti-mouse secondary antibody (1:200) and colorized with CF488A tyramide dye in amplification buffer. Without autofluorescence elimination, the sections were coverslipped using Fluoroshield with DAPI mounting solution (Sigma-Aldrich). To obtain the representative images, the neocortex and subcortical white matter was scanned using a Leica THUNDER Imager DMi8 under 10× lens, and the high-magnification images (63× lens) were also taken from the same regions.
7
Quantifying AHR and Iba-1 in Stroke
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Imaging was performed with Leica THUNDER Imager DMi8, using a 40x lens. Five images from both infarct and peri-infarct areas in stroke cases and five images from the normal cortical region in control cases were taken. The infarct and peri-infarct regions were confirmed using hematoxylin and eosin (H&E) staining by a neuropathologist. Images were analyzed using the ImageJ software by an investigator blinded to case conditions. All images were corrected for background noise and consistent fluorescence intensity. Quantities of AHR and Iba-1-positive cells were obtained using individual regions-of-interest (ROIs) within green or red-specific channels, respectively. The quantities of AHR/Iba-1 double-positive cells were measured using overlay images consisting of both channels, and counts were based on reselection of double-positive ROIs. Data are expressed as mean values ± SEM.
8
Brightfield and Immunofluorescent Imaging of ION
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All brightfield images were taken using a Leica Aperio LSM™ slide scanner under 20X objective. All immunofluorescent images were taken on the Leica Thunder imager DMi8. Images were acquired at 20x using a Leica K5 camera. Leica biosystems LAS X software was used for image capture. Tiles covering the entire ION were taken and stitched. Leica Thunder instant computational clearing was used to remove out of focus light. The images were exported as tiff files for downstream analysis.
9
Visualization of Surface Hsp70 Expression
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LS174T and UD‐5 cells (5 × 104 cells) were allowed to adhere to glass slides coated with poly‐L‐lysine (Sigma‐Aldrich) overnight. Cells were then incubated in a complete medium containing FITC‐conjugated cmHsp70.1 mAb for 30 min on ice, washed twice with PBS, and fixed with 4% w/v paraformaldehyde (PFA) in PBS for 15 min at RT. The cells were rewashed before being stained with phalloidin (1 µg mL−1) and DAPI (2 µg mL−1) for F‐actin and nuclei, respectively, for 1 h at RT in a light‐protected condition. Images were obtained using a fluorescence microscope with 63x oil objective (Leica, THUNDER Imager DMi8) equipped with Leica LAS X software.
10
Visualizing Nanoparticle Internalization in Tumor and Normal Cells
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To visualize the NP internalization, tumor (MDA-MB-231) and normal (PBL) cells were seeded overnight and then incubated in fresh complete medium containing FITC-labeled TPP-PEG4-FeAuNPs (2.5 µg/mL). Following a 24 h incubation period at 37 °C, cells were washed twice with cold PBS and then fixed with ice-cold 4% w/v paraformaldehyde (PFA) in PBS for 15 min at room temperature (RT). After an additional washing step, the filamentous actin (F-actin) and nuclei of the cells were stained with rhodamine-phalloidin (1 μg/mL) and DAPI (2 μg/mL), respectively, for 1 h at RT in the dark. Three-dimensional images of both the tumor (MDA-MB-231) and normal (PBL) cells were acquired using a fluorescence microscope with 63× objective (Leica, THUNDER Imager DMi8, Wetzlar, Germany) equipped with Leica LAS X 3.7.4 software (Leica, Wetzlar, Germany).
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