Liver specimens were fixed in 10% buffered formalin and embedded in paraffin. The sections were cut using a microtome (Leica SM 2000; Germany) and mounted on glass slides. Ten cross-sections from each liver were stained with Van Gieson picrofuchsin (VG) to analyze the general histoarchitecture of the tissues, the collagen structure, and histopathological changes. All sections were examined using light microscopy, on a Leica DM1000 system.
Sm 2000
Manufactured by Leica
Sourced in Germany
The SM 2000 is a stereo microscope designed for laboratory use. It provides high-quality optical performance and a wide range of magnification capabilities to facilitate detailed examination and analysis of samples. The core function of the SM 2000 is to enable precise and efficient observation and inspection of various specimens in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Dm1000 system, by Leica (1 mentions)
Dxm200f digital camera, by Nikon (1 mentions)
Streptavidin peroxidase, by Dianova (1 mentions)
Biotinylated anti mouse secondary antibody, by Agilent Technologies (1 mentions)
Fluoromount g with dapi, by Southern Biotech (1 mentions)
Ab11427, by Abcam (1 mentions)
S30 100ml, by Merck Group (1 mentions)
X100 100ml, by Merck Group (1 mentions)
Pt link, by Agilent Technologies (1 mentions)
Envision flex hrp system, by Agilent Technologies (1 mentions)
Las software program, by Leica (1 mentions)
Dm4000b, by Leica (1 mentions)
Fluoro gel mounting medium, by Electron Microscopy Sciences (1 mentions)
P6148, by Merck Group (1 mentions)
S32354, by Thermo Fisher Scientific (1 mentions)
A6455, by Thermo Fisher Scientific (1 mentions)
11 protocols using sm 2000
1
Histological Analysis of Liver Tissue
2
Immunohistochemical Analysis of Mouse Brain
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Mice used for immunohistochemistry were anesthetized by intraperitoneal pentobarbital injection (65 mg/kg), and subjected to intracardiac perfusion with cold PBS (pH 7.4) followed by 4% freshly prepared paraformaldehyde. Brains were immediately removed, post-fixed in the same fixative at 4°C overnight and then cryoprotected in 30% sucrose. Brains were then frozen and sectioned (40 μm) using a sliding microtome (Leica SM-2000). Free-floating sections were washed 3X in PBS buffer and blocked in PBS buffer containing 0.2% Triton-X and 3% normal donkey serum for 1 h at room temperature. The sections were incubated with primary antibodies overnight at 4°C followed by fluorophore-conjugated secondary antibodies (2 h, room temperature). Images were obtained in the Vanderbilt Cell Imaging Shared Resource using a Zeiss Axio Imager M2 microscope equipped with X-Cite 120 laser source (Lumen Dynamics). Confocal images were obtained using a Zeiss LSM 510 Meta confocal microscope.
3
Processing and Visualization of Implant Tissue Samples
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The tissue samples were processed to obtain non-decalcified ground sections. After 2 weeks in 10% neutral formalin, the specimens were rinsed in running tap water, trimmed, dehydrated in ascending concentrations of ethanol, and embedded in methyl methacrylate. The embedded tissue blocks were serially cut into 5-μm-thick ground sections using a microtome (Leica SM 2000; Germany) and mounted on glass slides. Ten cross-sections from the middle of each implant were stained with hematoxylin-eosin and Masson's trichrome and visualized using an optical microscope (DXM200F Digital Camera; Nikon, Tokyo, Japan).
4
Tissue Fixation and Histopathological Analysis
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The collected tissue samples from the right inferior lobe were fixed in 10% neutral formalin solution for 72 h at room temperature. The fixed tissues were examined under a light microscope, and paraffin blocks were formed. The prepared blocks were cut using a microtome (Leica SM 2000, Germany), and 4–5 μm thick sections were mounted on polylysine-coated slides. The sections were stained using hematoxylin–eosin to assess the structural changes, examined under light microscopy, and assessed in terms of histopathological parameters.
5
Histological Tissue Preparation Protocol
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The mice were sacrificed by cervical dislocation. Immediately after, samples of the pancreas, liver, kidney and gut were collected and fixed in 10% buffered formaldehyde (PH 7.2–7.4). Samples were further dehydrated by an ascending ethanol line (30%, 50%, 70%, 80%, 95%, 100%), lightened by xylene and fixed in paraffin. Paraffin blocks were cut using a microtome (Leica SM 2000, Prague, Czech Republic) into sections of 3.5 µm and stained by hematoxylin-eosine pigment.
6
Immunohistochemical Analysis of Fascin Expression
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Tissue sections of eight-micrometer thickness were taken from paraffin-embedded blocks using a standard microtome (Leica SM2000, Wetzlar, Germany) and mounted onto microscope slides for analysis. For heat-induced antigen retrieval, after dewaxing and rehydration, all slides were subjected to microwave treatment (3×5 min each, 600 W, in 10 mM citrate buffer, pH 6.0). The slides were then immersed into 3% H2O2 and 100% methanol in order to block endogenous peroxidase activity. Then, the slides were subjected to overnight incubation at 4 °C with mouse monoclonal anti-fascin antibody (1:50; Dako) and treated with biotinylated anti-mouse secondary antibody (1:100; DAKO). Finally, the slides were incubated with streptavidin-peroxidase (1:100; Dianova), and DAB/H2O2 (1.85 mM). PBS was used for all washing procedures. Haematoxylin was used for counterstaining the slides. As negative control, PBS was used instead of primary antibody. In addition, the endothelium tissues were considered as positive control of staining.
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Tissue sections of eight-micrometer thickness were taken from paraffin-embedded blocks using a standard microtome (Leica SM2000, Wetzlar, Germany) and mounted onto microscope slides for analysis. For heat-induced antigen retrieval, after dewaxing and rehydration, all slides were subjected to microwave treatment (3×5 min each, 600 W, in 10 mM citrate buffer, pH 6.0). The slides were then immersed into 3% H2O2 and 100% methanol in order to block endogenous peroxidase activity. Then, the slides were subjected to overnight incubation at 4 °C with mouse monoclonal anti-fascin antibody (1:50; Dako) and treated with biotinylated anti-mouse secondary antibody (1:100; DAKO). Finally, the slides were incubated with streptavidin-peroxidase (1:100; Dianova), and DAB/H2O2 (1.85 mM). PBS was used for all washing procedures. Haematoxylin was used for counterstaining the slides. As negative control, PBS was used instead of primary antibody. In addition, the endothelium tissues were considered as positive control of staining.
7
Immunohistochemical Analysis of Mouse Brains
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At the end of experiments, mice were transcardially perfused with 1x PBS followed by 4% paraformaldehyde solution. Brains were fixed overnight at 4 C, and then transferred to 30% sucrose solution for 48 hours. Brains were sectioned coronally using a microtome (Leica SM2000) at 50 um thickness and mounted on glass slides with Fluoromount G with DAPI (Southern Biotech, Birmingham, AL). Images were obtained using a confocal microscope (Nikon Ti2-E Crest LFOV Spinning Disk/ C2 Confocal) with 20X objective. Probe tracks were traced using the AllenCCF toolbox (https://github.com/cortex-lab/allenCCF ).
8
Perfusion and Immunostaining of Mouse Brains
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Mice P55–74 were anesthetized with a 4% isoflurane-oxygen mixture (v/v) and perfused transcardially with phosphate-buffered saline (PBS, Gibco, Life Science Technologies, 70011044) followed by 4% paraformaldehyde (PFA) in PBS. Extracted brains were further fixed in PFA for 24 h before being transferred to a 30% (w/v) sucrose solution and stored at 4°C. Brain slices were cut at 40 μm on a freezing stage sliding microtome (Leica SM2000) and stored in PBS. From each mouse, two brain slices were selected for immunostaining: one at approximately Bregma +1.98 mm and another at Bregma +0.98 mm along the anterior-posterior (A–P) axis. Slices were first blocked and made permeable in a solution containing 10% donkey serum (Sigma-Aldrich, S30–100ML) and 1% Tritonx100 (Sigma-Aldrich, X100–100ML) in PBS. Next, after applying the primary antibody (rabbit anti-PV, Abcam, ab11427) at a 1:250 dilution, slices were placed on a shaker at 4°C for 48 h. Slices were then washed 4 × 15 min with 0.025% Tritonx100 in PBS. Next, the secondary antibody was applied (anti-rabbit STAR RED, Abberior, STRED) at a 1:500 dilution and returned to the shaker for 48 h at 4°C. Slices were then washed 4 × 15 min in PBS, and mounted onto 1 mm microscope slides (Globe Scientific, #1324) with DAPI Fluoromount (Thermo Fisher Scientific, Cat. #: 00-4959-52).
9
Constructing Tissue Microarray for IHC
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All surgically removed specimens were fixed in 4% buffered formalin and routinely embedded in a low-melting paraffin. Paraffin blocks were used to construct tissue microarrays using a Manual Tissue Arrayer MTA 1 device (Beecher Instruments Inc, Sun Prairie, WI, USA). The hollow needle was used to extract tissue cores from donor blocks which were re-embedded into a single recipient block to form a microarray. Then, all blocks were cut into sections of 4 μm thickness on the microtome (Leica SM 2000, Nussloch, Germany). Before staining, the slides were incubated for 24 h at 37 °C, deparaffinized and rehydrated. For antigen retrieval, the heat-induced epitope retrieval method was used (PTLink, Dako, Glostrup, Denmark). For blocking, endogenous peroxidase slides were incubated in 3% H2O2 for 5 min. Manual staining procedure was conducted using the Dako EnVision Flex/HRP system (Glostrup, Denmark). Immunohistochemical staining was performed using rabbit Anti-ZNF-281 primary antibody (Sigma, HPA051228, 1:200, Saint Louis, MO, USA) (Figure 5 ).
10
Tissue Histology Processing and Analysis
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Tissue samples were collected and rinsed with sterile saline. After being xed in 10% neutral buffered formaldehyde for 72 hours, tissues were embedded in para n. Sections of 4 µm thickness were obtained from para n blocks on slides by using a microtome (Leica SM 2000, Germany). The sections were depara nized using xylene and rehydrated using a graded ethanol series. The rehydrated sections were then stained with hematoxylin and eosin (H&E), viewed under a computerized photo-light microscope (Leica DM4000B, Germany) and and LAS software program (Leica), coded for blind examination.
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