Brain paraffin sections (5 μm thick) were hydrated in a series of graded ethanol (96%, 85%, 70%, 50%) for 5 min each, washed in water and PBS, incubated with Hoechst 33258 (5 μg/mL) for 20 min. Images were visualized using a Leica DM5000 upright microscope (Leica Microsystems) at 20× magnification, Ex 460 nm/Em 490 nm at the excitation wavelength of 475 nm and emission wavelength of 555 nm.
Dm5000 upright microscope
Manufactured by Leica
Sourced in Germany
The Leica DM5000 is an upright microscope designed for use in research and industrial applications. The microscope features a modular design and supports a range of optical configurations, including brightfield, darkfield, and fluorescence microscopy. The DM5000 is equipped with high-quality optics and a sturdy, ergonomic body to provide reliable performance and user comfort.
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Lab products found in correlation
Anti rabbit cy3 conjugate secondary antibody, by Merck Group (3 mentions)
Mach1 kit, by Biocare Medical (1 mentions)
Anti phosphorylated nf κb, by Cell Signaling Technology (1 mentions)
Lab tek 2 chambered coverglass, by Thermo Fisher Scientific (1 mentions)
Fuchsin substrate chromogen system, by Agilent Technologies (1 mentions)
Lsab2 dako kit, by Agilent Technologies (1 mentions)
Anti phospho erk, by Santa Cruz Biotechnology (1 mentions)
Anti gfap, by Cell Signaling Technology (1 mentions)
Anti app, by Santa Cruz Biotechnology (1 mentions)
10 protocols using dm5000 upright microscope
1
Paraffin Brain Sections Visualization
2
Immunohistochemical Analysis of Muscle Fiber Types
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Muscles were fixed in a solution of acetone, methanol, and water (2:2:1) for 12 h, washed in tap water and dehydrated with ethanol at 70, 96, 100% v/v. After dehydration, the tissue pieces were placed in xylol for 1.5 h and embedded into paraffin. The embedded muscles were sliced into sections (5 μm) that were mounted on glass slides. For immunohistochemical analysis, the serial sections were incubated in an “antigen unmasking solution” (10 mM tri-sodium citrate, 0.05% Tween-20) for 10 min at 75 °C. Then, the MACH1 kit (M1u539 g, Biocare, Concord, CA, USA) was used according to the manufacturer's instructions. The sections were incubated in primary antibodies, including anti-myosin heavy chain-I (MHC-I, A4951, Hybridoma Bank, Iowa, IA, USA), anti-myosin heavy chain-IIa/IIx (MHC-IIa/IIx, A4.74, Hybridoma Bank, Iowa, IA, USA) or anti-myosin heavy chain-IIb (MHC-IIb, BF-F3, Hybridoma Bank, Iowa, IA, USA), in a humidified chamber overnight at 4 °C. The following day, the sections were incubated for 1 h with the secondary antibody and polymers as previously described [34 (link)]. Finally, the slides were cover-slipped, and images were captured with a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany). By examining serial cross-sections stained for MHC-I and MHC-IIA/X, it was also possible to identify type IIB fibers because of the negative staining.
3
Histological Examination of Salivary Gland
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Sections of salivary glands with a thickness of 5 μm were obtained from paraffin blocks and stained with hematoxylin and eosin (H&E) for histological examination. In brief, sections were de-waxed in xylene for 10 min and rehydrated by sequential immersion in decreasing ethanol concentrations. Then, the sections were stained with H&E [109 (link)] and analyzed using a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany). All sections were examined by two independent observers (F.C. and F.R.) in a blind manner, using coded slides and not knowing their source.
4
Phosphorylated NF-κB Immunostaining
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106/ml LAN5 cells were cultured on Lab-Tek II Chambered Coverglass (Nunc) and treated as described above. After washing in PBS, the cells were fixed in 4% paraformaldehyde for 30 min and stored at 4°C. After incubation with 3% BSA/PBS for 1 h, the cells were immunostained with anti-phosphorylated-NFκB (1 : 100; Cell Signaling) antibody at 4°C overnight. After washing in PBS, the samples were incubated with anti-rabbit Cy3-conjugate secondary antibody (1 : 500; Sigma). For nuclear staining, the cells were incubated with Hoechst 33258 (5 μg/ml) for 20 minutes. After washing, the cells were visualized by using a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany) at 20x magnification.
5
Histological Evaluation of Liver Injury and Fibrosis
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Liver tissue from the same hepatic lobe of all mice was excised from EtOH, EtOH-P, and Con mice, fixed in 10% buffered formalin, and embedded in paraffin as described previously.32 (link) Thin sections, obtained from paraffin blocks, were stained with hematoxylin–eosin for histological evaluation of liver injury and Masson's trichrome stain was performed for evaluation of liver fibrosis.33 (link) Slides were examined, and images captured under bright field with a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany). Liver sections were examined by two expert pathologists in a blind manner, using coded slides and not knowing their source. The mean of the results was used as data. Liver steatosis was classified using a semiquantitative scoring system divided into three fat grades (grade I=5–33% grade II=34–66% and grade III=67–100%).34 (link)
6
Immunohistochemical Analysis of CD4+ Cells
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Sections (5 μm thick) of paraffined-embedded brains were hydrated in a sequence of graded ethanol (from 96% to 50%) for 5 min each, washed in water and then PBS. The slides were incubated with 3% BSA for 1 h and subsequently, with anti-CD4+ (1:40) (Dako) at 4 °C overnight. After washing, the LSAB2 Dako Kit (Dako) and Fuchsin Substrate-Chromogen System (Dako) were used for brown staining. The slides were mounted with cover slips, and images were visualized using a Leica DM5000 upright microscope (Leica Microsystems) at a magnification of 20×.
7
Thioflavin-T Staining of Brain Sections
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For Thioflavin-T (ThT) staining the brain sections including the cerebral cortex and the hippocampus were mounted on slides The slides were deparaffinized in xylene solution and hydrated in a series of graded ethanol (96%, 85%, 70%, 50%) for 5 minutes each. After washing in water, the sections were incubated in filtered 1% aqueous ThT solution for 8 minutes at room temperature. The slides were then dehydrated in ethanol 80% and 95%, for 5 minutes each. After washing in water the slides were mounted with cover slips and the images were visualized by using a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany) at 20x magnification.
8
Histological Analysis of Testes
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After the killing of animals, samples of testes were taken from each mouse for histological analysis as described previously 18 , 19 . Sections were stained with haematoxylin and eosin, mounted with coverslips and finally observed with a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany). Two independent observers (F.C and F.R) examined the specimens on two separate occasions, in a blind manner, using coded slides without knowing their source.
For the histological evaluation, 10 sections which had a 20‐μm distance from each other were observed with the light microscopy and the images were taken at ×40 magnification.
For the histological evaluation, 10 sections which had a 20‐μm distance from each other were observed with the light microscopy and the images were taken at ×40 magnification.
9
Immunofluorescence Imaging of Brain Sections
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For immunofluorescence, the brain was embedded in paraffin as previously described [14 (link)] and coronally sectioned (5 μm) using a microtome. Brain sections, including the cerebral cortex, corpus callosum, hippocampus, thalamus and hypothalamus, were mounted on slides and deparaffinized in xylene solution. Then, the slides were hydrated in a series of graded ethanol (96%, 85%, 70%, 50%) for 5 min each. After washing in water and PBS, the slides were incubated with 3% BSA/PBS for 1 h. Next, the sections were incubated with anti-phospho-ERK (1:50, Santa Cruz Biotechnology) and anti-GFAP (1:50, Cell Signaling Technology), respectively, at 4 °C overnight. After washing in PBS, the samples were incubated with anti-rabbit Cy3-conjugate secondary antibody (1:500; SIGMA). After washing in PBS, the slides were mounted with cover slips, and the images were visualized using a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany) at 20× magnification.
10
Immunofluorescence Analysis of APP in Brain
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For immunofluorescence the brains (n=5 per group) were embedded in paraffin as previously described [7 (link)] and coronally sectioned (5μm) using a microtome. Brain sections including the cerebral cortex and the hippocampus were mounted on slides and deparaffinized in xylene solution. Then, the slides were hydrated in a series of graded ethanol (96%, 85%, 70%, 50%) for 5 minutes each. After washing in water and PBS the slides were incubated with 3% BSA/PBS for 1 h. Next, the sections were incubated with anti-APP (1:50) (Santa Cruz) at 4°C overnight. After washing in PBS, the samples were incubated with anti-rabbit Cy3-conjugate secondary antibody (1:500; SIGMA). For nuclear staining, the sections were incubated with Hoechst 33258 (5μg/ml) for 20 minutes. After washing in PBS the slides were mounted with cover slips and images were visualized by using a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany) at 20X magnification.
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