Histological examination of the liver was done after staining sections with hematoxylin and eosin (H&E). After fixation in 10% neutral formaldehyde, liver specimens were dehydrated, wrapped, cut into sections, and stained with H&E solution. Lipid deposition in the liver was assessed by staining with Oil Red O. Briefly, cryostat sections (8 μm) of the liver were fixed, stained with Oil Red O (Sigma-Aldrich, St. Louis, MO, United states) solution, and then counterstained with hematoxylin (Sigma-Aldrich). The sections were examined under the Leica DM4000B light microscope.
Dm4000b light microscope
Manufactured by Leica camera
The Leica DM4000B is a light microscope designed for laboratory use. It features a high-quality optical system that provides clear and detailed images of specimens. The microscope is suitable for a range of applications and can be configured with various accessories to meet specific needs.
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Lab products found in correlation
Hematoxylin, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Ultracut r, by Leica camera (1 mentions)
Safranin o, by Merck Group (1 mentions)
Formalin solution, by CellPath (1 mentions)
Masson s trichrome, by Merck Group (1 mentions)
Dm 5000b fluorescence microscope, by Leica camera (1 mentions)
Mab377b, by Merck Group (1 mentions)
Vectastain elite abc rabbit igg kit, by Vector Laboratories (1 mentions)
Openlab, by PerkinElmer (1 mentions)
A0564, by Agilent Technologies (1 mentions)
Goat anti rabbit, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
11 protocols using dm4000b light microscope
1
Histopathological analysis of liver lipid deposition
2
Analysis of Cambium-Derived Tissue in Transgenic Plants
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The second internode, 10th internode, and 20th internode (counting from the tip) were taken from 4-month-old wild-type and 35S::PtoCYCD3;3 plants. The samples were fixed in FAA (70% ethanol: glacial acetic acid: formaldehyde: glycerol = 18:1:1:1) for 24 h at room temperature and then dehydrated through a graded ethanol series (70%, 80%, 90%, and 100% ethanol; 20 min each step). Ethanol was replaced with ethanol: acetone (1:1) for 20 min and then with pure acetone for 20 min two times. Afterward, the samples were sequentially infiltrated with acetone: resin (2:1) for 3 h and then with acetone: resin (1:1) and acetone: resin (1:3) for 3 h. The samples were replaced with fresh resin for 12 h. After the resin replacement was repeated two times, stem segments were embedded into the resin for 24 h at 60 °C. One µm sections were obtained with a microtome (Leica ultracut R, Germany). The sections were floated on the water, heat-fixed to glass slides, stained with 0.05% toluidine blue, and observed under a Leica DM4000B light microscope. Data were measured on Image J. We measured five times in different quadrant sectors of the cross-sections about the width of cambium-derived tissues. All statistical analyses were performed using SPSS v16.0. Independent-Samples T-test was used.
3
Histological Analysis of Day 7 Cartilage
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Due to their larger size, histology was performed only on the day 7 cartilage discs. One half of a disc was sectioned, fixed in 10% formalin solution (CellPath) overnight and wax embedded following standard processing protocols. Four μm transverse sections were taken, with one section from each treatment mounted onto each Cellstik Superfrost Ultra Plus slide (CellPath). Slides were stained with haematoxylin and eosin (Licor), Safranin-O (Sigma) and Masson’s Trichrome (Sigma) and images were taken using a Leica DM4000B light microscope at 20 x magnification.
4
Immunohistochemical Tissue Analysis
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Five µm thick formalin-fixed paraffin-embedded tissue were deparaffinized, rehydrated and incubated in 0.3% (w/v) H2O2 in dH2O for 30 min to block endogenous peroxidase activity. Heat-induced antigen retrieval was carried out in 10 mM citrate buffer (pH 6.0) or Tris/EDTA buffer (pH 9.0). Tissue was then stained according to standard protocols using the antibodies listed in Table 2 . Immunopositivity was detected using 3,3′-diaminobenzidine chromogen or chromogen-bound Alexa Fluor®-labelled secondary antibodies. Hematoxylin or DAPI-Fluoromount-G® (ITK diagnostics, Uithoorn, The Netherlands) were used as counterstains. Images were taken using a Leica DM4000B light microscope or a Leica DM5000B fluorescence microscope.
5
Quantifying Hippocampal Neuron Counts
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Tissue was stained with biotinylated NeuN (1:100, EMD Millipore MAB377B) as described above, but without 0.5% nickel sulfate hexahydrate and TBS. Tissues were mounted and before coverslipping they were counterstained with 0.05% cresyl violet (Nissl) to quantify neurons. Only those positive for both NeuN and Nissl stains in the Cornu ammonis 1 (CA1) region of the hippocampus were counted. A stereology workstation consisting of a modified Leica DM4000B light microscope with a Prior motorized stage was used to outline the area using distinct landmarks in the brain at 4× magnification [2 (link), 69 (link)]. Neurons in this region were counted using randomly designated areas in the computer-generated grid using a 100 × oil immersion objective. Neurons were counted when they were located within the three-dimensional dissectors or touching the inclusion lines, and the top 1 µm and bottom 1 µm of tissue were excluded. High magnification images of tau staining in the hippocampus were also taken using the high power (100x, NA 1.3) oil immersion objective on this microscope.
6
Corneal Morphology in PM10 Exposure
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Whole eyes (n = 3) were harvested from mice after 2 weeks of exposure to control or 500 μg/m3 PM10. Eyes were fixed in alcoholic z-fix and sent to Excalibur Pathology, Inc (Norman, OK) where they were embedded in paraffin, sectioned and stained with H&E. Photographs of representative areas of the epithelium were taken using a Leica DM4000B light microscope at 20X. The thickness of the whole cornea, epithelium and stroma were analyzed from three mice in each group (72 measurements/group/treatment). Data are represented as thickness (in mm) and expressed as mean + SD.
7
Quantification of IgE and Tryptase in Skin and Blood
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A rabbit polyclonal antibody against human IgE was purchased from Thermo Fisher Scientific (Catalog number PA5-16396, MA, USA). The rabbit polyclonal antibody against purified human skin tryptase is an in-house antibody produced previously (30).
The skin samples were processed for 5-µm sections followed by fixation in 10% formalin and immunohistochemical staining using a 1:700 dilution of anti-IgE or 0.183 µg/ml anti-tryptase antibody. The immunopositive cells were visualized using a Vectastain Elite ABC Rabbit IgG Kit (Vector PK-6101, Vector Laboratories, CA, USA). The cells immunopositive for IgE or tryptase were counted in separate sections from an area of 1.0 mm (width) × 0.6 mm (depth) immediately beneath the epidermis by using an ocular grid (31,32). All samples were analyzed with Leica DM 4000B light microscope equipped with a 40x Plan Leica objective.
The blood samples were taken from the cubital fossa vein from 381 subjects. Blood tests included complete blood cell count, serum tryptase and serum total IgE. IgE was analyzed with electrochemiluminescence immunoassay and serum tryptase with ImmunoCAP TM assay (20,24).
The skin samples were processed for 5-µm sections followed by fixation in 10% formalin and immunohistochemical staining using a 1:700 dilution of anti-IgE or 0.183 µg/ml anti-tryptase antibody. The immunopositive cells were visualized using a Vectastain Elite ABC Rabbit IgG Kit (Vector PK-6101, Vector Laboratories, CA, USA). The cells immunopositive for IgE or tryptase were counted in separate sections from an area of 1.0 mm (width) × 0.6 mm (depth) immediately beneath the epidermis by using an ocular grid (31,32). All samples were analyzed with Leica DM 4000B light microscope equipped with a 40x Plan Leica objective.
The blood samples were taken from the cubital fossa vein from 381 subjects. Blood tests included complete blood cell count, serum tryptase and serum total IgE. IgE was analyzed with electrochemiluminescence immunoassay and serum tryptase with ImmunoCAP TM assay (20,24).
8
Brain Tissue Cryosectioning and Imaging
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Brain tissues were cryosectioned to 8 μm thicknesses using a Microm HM 550 cryostat, and then mounted onto glass slides and stained with H & E. Brain tissue sections were imaged using a Leica DM4000B light microscope fitted with a 100x/1.25NA, 50x/0.9NA, 20x/0.4NA or 10x/0.25NA N Plan objective lens. Images were captured by a MicroPublisher 3.3RTV camera (QImaging) controlled by OpenLab software (Improvision/PerkinElmer). Regional image capture and analysis were undertaken for all control and alcoholic samples, for which representative images from a control and alcoholic matched pair are included as Figure 1 .
9
Murine Pancreas Histology and Immunohistochemistry
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FFPE murine pancreas tissue cut into 5-μm sections was used for histological and immunohistochemical analysis. VP1 was detected with a biotinylated anti-VP1 antibody (5D8/1, Dako; biotinylated by Capra Bioscience, Ängelholm, Sweden; final concentration, 0.07 μg/ml) and tris-EDTA (pH 6) antigen retrieval. Biotin was then detected using the Vectastain ABC Elite Biotin System (Vector Laboratories) and stained using DAB (Vector Laboratories; both according to the manufacturer’s instructions). As previously described, the antibody was validated in mouse pancreas sections (20 (link), 27 (link), 32 (link)). The islet hormones insulin and glucagon were detected with anti-insulin (1:10,000; A0564, Dako) and anti-glucagon (1:12,000; AB92518, Abcam) antibodies. Secondary antibodies alone were used as negative controls in the hormone staining (insulin: goat anti-guinea pig, Vector Laboratories; glucagon: goat anti-rabbit, Dako) or 2% normal goat serum the case of the VP1 staining. The images were acquired on a Leica DM4000B light microscope using QWIN software.
10
Cytological Analysis of Root Tips
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The root tips were cut off and fixed in ethanol-acetic acid (3 : 1) solution for at least 24 h at 4 C. The root tips were hydrolyzed in 1 M HCI at 60 C for 12-13 min and then rinsed with tap water for 2-3 min (Inceer et al. 2002) . Staining was carried out in Shiff-Reagent for 1.5 h. For each variable, 5-6 root tips squashes were prepared, and a minimum of 500 mitotic cells were counted from each slides under a light microscope (Leica DM 4000B light microscope).
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