After 24 h fixation, the ileum segment was dehydrated with gradient alcohol, made transparent with xylene, and embedded in paraffin. To determine the effects of pectin supplementation on the LPS challenge-induced damage to intestinal integrity, we evaluated the ileal morphology using hematoxylin and eosin (H&E staining kit, Solarbio, Beijing, China) staining. Five cross-sections (5 µm thick) of each ileum specimen were taken to prepare histological sections and stained, then observed on a Leica DM20 0 0 light microscope (Leica Microsystems, Wetzlar, Germany). The images were analyzed with ImageJ v1.8.0 software. Fifteen well-oriented and intact villi and their associated crypts were taken from each segment. Villus height (VH), crypt depth (CD), and the ratio of villus height to crypt depth (VH:CD) were measured. Goblet cells were examined by periodic acid-Schiff-Alcian Blue stain (PAS-AB staining kits, Solarbio, Beijing, China). Briefly, deparaffinized slides were exposed to 3% acetic acid, followed by staining with Alcian Blue, 1% periodic acid-Shiff reagent, and sodium metabisulfite. Finally observed and analyzed on a Leica DM20 0 0 light microscope (Leica Microsystems, Wetzlar, Germany).
Dm2000 light microscope
Manufactured by Leica
Sourced in Germany
The Leica DM2000 is a light microscope designed for general laboratory use. It features a binocular viewing head, allowing for comfortable observation. The microscope provides bright, high-contrast images through its optical system. It is intended for a variety of routine applications in research and educational settings.
Automatically generated - may contain errors
Lab products found in correlation
Dfc425c, by Leica (2 mentions)
Application suite v3, by Leica (2 mentions)
Rm2255 microtome, by Leica (2 mentions)
Scn400 slide scanner, by Leica (2 mentions)
Entellan, by Merck Group (2 mentions)
Rm2125 microtome, by Leica (2 mentions)
Oil red o working solution, by Wuhan Servicebio Technology (1 mentions)
Bond max, by Leica (1 mentions)
Giemsa, by Avantor (1 mentions)
Facscan, by BD (1 mentions)
Microtome, by Thermo Fisher Scientific (1 mentions)
Collagen 1, by Merck Group (1 mentions)
Collagen 3, by Merck Group (1 mentions)
He staining kit, by Solarbio (1 mentions)
Real envision peroxidase dab detection system, by Agilent Technologies (1 mentions)
Rm2245 rotary microtome, by Leica (1 mentions)
Paraplast plus, by Merck Group (1 mentions)
Dig rna labeling kit, by Roche (1 mentions)
Dfc295 digital camera, by Leica (1 mentions)
Ultracut uct, by Leica (1 mentions)
39 protocols using dm2000 light microscope
1
Histological Assessment of Intestinal Integrity
2
Intestinal Histomorphometry: Detailed Protocol
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Histo-morphological measurements were performed as previously described (Metzler-Zebeli et al., 2018b (link)). After fixation, intestinal tube pieces were dehydrated in ethanol, cleared in xylene and embedded in paraffin. Three discontinuous 3–4 μm-thick sections per intestinal site were routinely stained with hematoxylin and eosin. The Leica DM2000 light microscope (Leica Microsystems, Wetzlar, Germany) fitted with a digital camera (Leica DFC425C) and the Leica Application Suite V3.7 software was used to take pictures which were analyzed with ImageJ software (Version 1.47; National Institutes of Health, Maryland, United States). Fifteen intact well-oriented, crypt-villus units were selected, with the criterion for villus selection based on the presence of intact lamina propria. Villus height and width were measured at 4-times magnification and crypt depth at 10-times magnification. The circular and longitudinal muscular layers were measured. Goblet cells were counted per 250 μm of villus or crypt epithelium, 15 replicates per gut site at 10-times magnification, whereas intraepithelial lymphocytes were counted per 400 μm villus epithelium, 12 replicates per gut site at 20-times magnification. Goblet cell counts and intraepithelial lymphocytes were presented per villus-crypt unit.
3
Histological Assessment of Liver Tissues
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Liver sections were submerged in 4% paraformaldehyde for 24 h, followed with a standard hematoxylin and eosin (H&E) staining procedure. Oil red O staining were performed in liver tissues frozen in OCT and stained in oil red O working solution (Servicebio technology, Wuhan, China). All slides were photographed using a Leica DM2000 light microscope (Leica Microsystems, Inc.; magnification, x100 or x200).
4
Determination of Malaria Parasitemia and Reticulocyte Infection
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A blood smear was taken at indicated time points and stained with Giemsa (1/10 dilution, VWR, Heverlee, Belgium) to obtain percentages of parasitaemia, reticulocytes and infected reticulocytes. Additionally, 1/500 diluted tail blood was counted using a Bürker chamber to obtain RBC concentrations, from which numbers of iRBCs, reticulocytes and infected reticulocytes per ml were calculated. Images of Giemsa-stained blood smears were taken at a magnification of 100 (100×/1.25 oil objective) with a Leica DM 2000 light microscope equipped with a DFC 295 camera (Leica Bond Max, Leica Microsystems, Diegem, Belgium).
For determination of parasitaemia by flow cytometry [35 (link)], tail blood (10 µl) from infected mice was collected in 1 ml of complete culture medium and cells were fixed in 1 ml of a 0.25% (v/v) glutaraldehyde solution in PBS at 4 °C and kept at 4 °C until analysis. These samples were stained with the DNA-specific fluorescent dye Hoechst-33258 (2 µmol/l) for 1 h at 37 °C and analysed with a a FACScan (LSR II, Becton–Dickinson). The fluorescence intensity and size (Forward Scatter; FSC; Sideward Scatter, SSC) of 50,000 cells per sample were measured and data analysis was performed using the FlowJo software (FlowJo, LLC, Ashland, USA).
For determination of parasitaemia by flow cytometry [35 (link)], tail blood (10 µl) from infected mice was collected in 1 ml of complete culture medium and cells were fixed in 1 ml of a 0.25% (v/v) glutaraldehyde solution in PBS at 4 °C and kept at 4 °C until analysis. These samples were stained with the DNA-specific fluorescent dye Hoechst-33258 (2 µmol/l) for 1 h at 37 °C and analysed with a a FACScan (LSR II, Becton–Dickinson). The fluorescence intensity and size (Forward Scatter; FSC; Sideward Scatter, SSC) of 50,000 cells per sample were measured and data analysis was performed using the FlowJo software (FlowJo, LLC, Ashland, USA).
5
Hematoxylin and Eosin Staining of Mouse Liver
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The control group and the experimental group model mice were injected with pcDNA3.1-HBx, empty pcDNA3.1 plasmids and plasmid solvent solution into the tail vein for 24 h, and liver tissues sized 1.0×1.0×0.3 cm were obtained, fixed with 4% paraformaldehyde for 24 h at room temperature. Subsequently, the tissue sections were dehydrated in 80, 90, 95 and 100% gradient ethanol for 2–4 h. The embedded liver tissue was cut into 3-µm sections, using the microtome (Thermo Fisher Scientific, Inc.), and dried for H&E staining. Following 20 min of baking at 60°C, xylene I and II were used to dewax the tissues for 10 min each. Subsequently, tissues were incubated in 100, 95, 85, 75% gradient ethanol for 5 min each, and in distilled water for 5 min to complete the dewaxing process. Hematoxylin (cat. no. C0390; Beijing Noblelight Technology Co., Ltd.) staining was performed for 5 min at room temperature, after washing with water. Eosin staining (cat. no. C0390, NobleRyder) was conducted for 5 sec, and the sections were subsequently fully washed (33 (link)). Sections were then dehydrated using 75, 85, 95 and 100% ethanol (2 min each). Xylene I, II transparent for 5 min neutral gum seal is intended for fixation. Sections were observed and images were captured using a Leica DM2000 light microscope (Leica Microsystems, Inc.; magnification, ×20).
6
Assessing Pubertal Maturation in Mice
7
Histological Analysis of Ileum and Cecum
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The ileum and cecum samples were dehydrated, embedded in paraffin, and then sliced at 5 μm thickness. The samples were stained with hematoxylin and eosin (H&E) and observed by a Leica DM2000 light microscope (Leica Microsystems, Wetzlar, Germany). The images were analyzed with Image J v1.8.0 software. Eight replicates of complete villus and crypt from each histological section were selected for measurement, and at least 4 vision fields were chosen.
8
Quantitative Analysis of Collagen I and III
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Frozen LV were embedded in Tissue-Tek (Sakura, Staufen, Germany) for cryosectioning and subsequent staining. As described previously51 (link), specific antibodies directed against Collagen I (Chemicon, Limburg/Lahn, Germany) and Collagen III (Merck Millipore, Darmstadt, Germany) were used. Quantitative analysis of the positive area (%)/heart area (HA; mm2) was performed by digital image analysis on a Leica DM2000 light microscope (Leica Microsystems, Wetzlar, Germany) at 100× magnification.
9
Intestinal Histomorphology Analysis via H&E
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Hematoxylin and eosin (H&E) staining was used to analyze intestinal histomorphology including villus height, crypt depth, and V/C ratio cited as described by Yin et al. [24] (link). The duodenum and jejunum samples were dehydrated, embedded in paraffin, and then sliced at 5 µm thickness. The samples were stained with H&E and observed on a Leica DM2000 light microscope (Leica Microsystems, Wetzlar, Germany). The images were analyzed with ImageJ version 1.8 software (National Institutes of Health, MD, USA). Two replicates of complete villus and crypt from each histological section were selected for measurement.
10
Histological Evaluation of Colonic Anastomosis
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A 1 cm-long sample including colonic anastomosis was immediately fixed in 4% formaldehyde. The samples were dehydrated, embedded in paraffin and cut in 4-µm thick slices. Histopathologic sections were stained with hematoxylin-eosin (HE) for inflammation parameters and picrosirius red for collagen. A conventional binocular Leica DM 2000 light microscope (Leica Microsystems, Wetzlar, Germany) was used for analysis. The samples were assessed in a blinded manner by two pathologists to avoid bias.
Inflammation parameters including necrosis, polymorphonuclear cells, lymphocytes, macrophages, edema, state of epithelial layer, and bridging of submucosal-muscular layer were scored according to Verhofstadt scale19 (link). Collagen formation was assessed as decreased deposition (0), normal (1) or increased deposition (2).
Inflammation parameters including necrosis, polymorphonuclear cells, lymphocytes, macrophages, edema, state of epithelial layer, and bridging of submucosal-muscular layer were scored according to Verhofstadt scale19 (link). Collagen formation was assessed as decreased deposition (0), normal (1) or increased deposition (2).
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