After radiological analysis, samples were fixed in 4% PFA for 48 hours, then decalcified in 10% ethylenediamine tetraacetic acid (EDTA; Leagene) for 6 weeks. To perform H&E and immunohistochemistry staining, samples were embedded in paraffin and cut into 5 μm sections. H&E staining was used to detect empty lacuna under light microscopy (Leica). Immunohistochemistry staining was also performed to examine osteogenic, adipogenic and angiogenic differentiation using, osteocalcin (OCN; GeneTex), adiponectin (GeneTex) and CD31 (GeneTex), respectively. Briefly, sections were fixed and stained with primary antibodies, followed by incubation with secondary antibodies (Abcam) conjugated to horse radish peroxidase (HRP). Peroxidase activity was visualized with diaminobenzidine (Sigma). Immunohistochemical images were observed using light microscopy (Leica). The histology and immunohistochemistry region for analysis is shown in Figure S1 .
Light microscopy
Manufactured by Leica
Sourced in Germany, United States, Japan, United Kingdom
Light microscopy is an optical imaging technique that uses visible light and a system of lenses to magnify small objects. It allows for the observation and study of microscopic samples, such as biological cells, tissues, and materials. The core function of light microscopy is to provide a detailed, high-resolution view of these small-scale structures.
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Lab products found in correlation
Microtome, by Leica (6 mentions)
Hematoxylin, by Merck Group (4 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (3 mentions)
Paraffin, by Merck Group (3 mentions)
Alcian blue, by Merck Group (3 mentions)
Fluorescence microscope, by Leica (2 mentions)
B900780, by Proteintech (2 mentions)
Anti mouse rabbit universal immunohistochemical detection kit pk10006, by Proteintech (2 mentions)
Rm2125, by Leica (2 mentions)
Hexion and e staining kit, by Bio-Rad (2 mentions)
Aquatex, by Merck Group (2 mentions)
Oil red o, by Merck Group (2 mentions)
Image pro plus software, by Leica (2 mentions)
Moticam pro 252b, by Nikon (2 mentions)
Jsm 6390 lv scanning electron microscopy, by JEOL (2 mentions)
Mowiol, by Leica (2 mentions)
Transwell chamber, by Corning (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Microsystems microtome, by Leica (2 mentions)
Ds ri1 camera, by Nikon (2 mentions)
192 protocols using light microscopy
1
Histological and Immunohistochemical Analysis of Cellular Differentiation
2
Histopathological Assessment of Liver Tissues
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The histopathological studies were made based on prior published methods (Ginsberg et al., 1981 (link); Al-Griw et al., 2015 ). Following dissection, 10% neutral buffered formalinfixed tissues were dehydrated in a series of increasing concentrations of ethanol solutions, cleared in xylene, and then embedded in paraffin wax. Paraffin sections were cut at 5 µm thick, deparaffinized, hydrated, stained with H&E, and examined under light microscopy (Leica, Germany). The hepatic tissue architecture was examined and imaged using light microscopy (Leica, Germany). The tissues from each animal were assessed blindly by two pathologists for histopathological changes.
Histopathologic investigation of liver sections was performed with particular consideration for inflammatory changes, hepatocellular necrotic changes, biliary ductless changes, fibrosis, architectural changes, and degenerative changes. The frequency of lesions was unbiasedly evaluated to determine a pattern of injury, which was assessed semi-quantitatively according to the severity and distribution of the pathological changes. Note that the only diagnoses that were considered to be accurate based on histopathological changes and data presented in each group were included in the histopathological description and depictions.
Histopathologic investigation of liver sections was performed with particular consideration for inflammatory changes, hepatocellular necrotic changes, biliary ductless changes, fibrosis, architectural changes, and degenerative changes. The frequency of lesions was unbiasedly evaluated to determine a pattern of injury, which was assessed semi-quantitatively according to the severity and distribution of the pathological changes. Note that the only diagnoses that were considered to be accurate based on histopathological changes and data presented in each group were included in the histopathological description and depictions.
3
Quantitative Lipid Staining in Cells and Tissue
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Mature 3T3-L1 adipocytes were washed once with phosphate-buffered saline (PBS), fixed in 4% solution of formaldehyde in PBS for 30 min, and stained with Oil Red O working solution (0.3% w/v Oil Red O in 60% isopropanol) for 40 min at room temperature (RT). The cells were then washed five times with distilled water. The signal was detected by light microscopy (Leica Microsystems, Mannheim, Germany). For quantification, Oil Red O was extracted into 100% isopropanol for 10 min and absorbance read at 500 nm.
Frozen liver tissue was cut using the microtome into 10-µm slices and fixed in 4% formaldehyde in PBS for 10 min at RT. The tissue was washed with water and distilled water, and rinsed in 60% isopropanol for 5 min. Lipids in the tissue were stained with Oil Red O working solution (see above) for 20 min at RT and washed in 60% isopropanol for 5 min and distilled water for 5 min. The nuclei were stained with hematoxylin for 5 min, and the tissue was washed twice with distilled water for 5 min each. Coverslips were mounted in Mowiol and the signal detected by light microscopy (Leica Microsystems) and evaluated using the Fiji software.
Frozen liver tissue was cut using the microtome into 10-µm slices and fixed in 4% formaldehyde in PBS for 10 min at RT. The tissue was washed with water and distilled water, and rinsed in 60% isopropanol for 5 min. Lipids in the tissue were stained with Oil Red O working solution (see above) for 20 min at RT and washed in 60% isopropanol for 5 min and distilled water for 5 min. The nuclei were stained with hematoxylin for 5 min, and the tissue was washed twice with distilled water for 5 min each. Coverslips were mounted in Mowiol and the signal detected by light microscopy (Leica Microsystems) and evaluated using the Fiji software.
4
Colony Formation Assay Protocol
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The colony formation assay was performed as described previously [24 (link)]. Briefly, 1 × 103 SiHa or CaSki cells were plated in each well of a 24-well plate (3524; Corning) with 3 replicates. After culture for 7 days, the cells were washed with PBS and fixed with 100% methanol for 1 h, stained with 0.5% crystal violet and imaged via light microscopy (Leica, Germany) to determine the number of cells.
5
Pancreas Histological Analysis Protocol
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The pancrease was fixed in 10% neutral formalin for 48 h, after being dehydrated in graded alcohol, transparent in xylene, then embedded in paraffin wax. Tissue sections of 5-μm thickness were sliced and routinely stained with hematoxylin–eosin (HE). Histological differences between the groups were viewed and photographed with light microscopy (Leica, Wetzlar, Germany).
6
BMSC-sEVs Promote HUVEC Migration
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HUVECs (1 × 104 cells/well) were resuspended in serum-free medium and seeded into the upper chamber of Corning 8-μm pore size transwell units (Corning, NY, United States) that were housed in 24-well plates. The lower chamber was filled with DMEM supplemented with 10% sEV-depleted FBS that was pre-incubated with PBS (200 μL), BMSC-sEVs (50 μg/ml), or BMSC-sEVs (100 μg/ml). The plates were incubated at 37°C for 24 h. Thereafter, the cells attached to the upper surface of the filter membranes were removed, and those attached to the lower surface were stained with 0.1% crystal violet. Cell migration was observed by light microscopy (Leica, Solms, Germany).
7
Histological Evaluation of Myocardial Damage
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The Paraffin-embedded slices were stained with hematoxylin-eosin (HE) for morphologic examination. The extent of myocardial damage was evaluated in 200-fold magnification by light microscopy (Leica, Germany).
8
Histological Analysis of Renal Fibrosis
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Portions of renal tissue fixed in 4% paraformaldehyde were embedded in paraffin and sliced into 4 μm-thick sections. The sections were stained with HE for assessment under light microscopy (Leica, Wetzlar, Germany). Renal sections 4 μm thick were stained with Sirius red to evaluate the area occupied by collagen fibrils. From each kidney, ten random interstitial cortical fields were captured at 40× magnification using a high-resolution video camera (Leica) connected to a light microscope (Leica DM 300 LED). The area occupied by collagen was measured using a computerized image analysis system (Image-Pro Plus).
9
Morphology and Growth of D. salina Transformant
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The cell morphology of the wild-type and DsLCYB-oe1-transformed strain was observed by light microscopy (Leica, Germany). Finally, we studied the effect of the DsLCYB-oe1 transformant on the growth of D. salina. The initial number of algal cells in each experimental group was the same, with culture growth monitored at regular intervals under different light conditions for 14 days by measuring the optical density at OD654 using a UV-visible spectrophotometer (UV 2550, Shimadzu).
10
Histological Evaluation of Lung Tissues
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The lung tissues isolated from different mice were fixed with 10% neutral phosphate-buffered formalin overnight, and embedded in paraffin. After that, sections were prepared and stained with hematoxylin/eosin for histological evaluation by light microscopy (Leica, Germany).
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