Deparaffinized 3 μm thick sections were pretreated for 20 min with 0.2 M HCl, incubated with 1 M NaSCN for 30 min at 80°C, washed in dH2O and 2x SCC and digested with 1 mg/ml pepsin (2,500–3,500 U/mg, Sigma Chemical, St. Louis, MO) in 0.14 M NaCl solution, pH2. The biotin labeled full length MCPyV DNA probe was added to the samples at a concentration of 5 ng/μl followed by denaturation of DNA (5 min, 80°C) and hybridization overnight (37°C, humid chamber, Thermobrite, Abbott, IL). Unbound MCPyV DNA probe was stringently washed away in 2x SSC, pH7 at 70°C for 2 min. Bound probe was detected by sequential incubation in a combination of fluorescein isothiocyanate (FITC) biotinylated avidin (AvFITC; 1:500; Vector, Brunswig Chemie, Amsterdam, The Netherlands) and biotin conjugated goat antiavidin (BioGaA; 1:100; Vector). Prior to incubation aspecific binding sites were blocked with Boehringer Blocking reagent. Cell nuclei were counterstained and coverslipped with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; 0.2 μg/ml, Vectashield, Vector Laboratories, CA). Samples were visualized using a DM 5000B fluorescence microscope (Leica, Wetzlar, Germany) coupled to an online digital camera (Leica DC 300 Fx) for independent evaluation of FISH signals by 3 investigators (AzH, DR, LH) according to criteria described earlier (Hafkamp et al., 2008 (link); Haugg et al., 2014 (link)).
Dc 300 fx
Manufactured by Leica
Sourced in Germany
The DC 300 Fx is a digital camera module designed for use in laboratory equipment. It provides high-resolution image capturing capabilities for various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Pepsin, by Merck Group (2 mentions)
Dm 5000b fluorescence microscope, by Leica (2 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Vector Laboratories (2 mentions)
Thermobrite, by Abbott (1 mentions)
Boehringer blocking reagent, by Roche (1 mentions)
Thermobrite system, by Abbott (1 mentions)
Cacl2 2h2o, by Merck Group (1 mentions)
Mgso4 7h2o, by Merck Group (1 mentions)
A7085, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Methylcellulose, by Merck Group (1 mentions)
Mz apo stereomicroscope, by Leica (1 mentions)
Ms 220, by Merck Group (1 mentions)
Dmlb 80 microscope, by Leica (1 mentions)
Qwin software, by Leica (1 mentions)
Paraplast, by Merck Group (1 mentions)
5 protocols using dc 300 fx
1
FISH-Based Detection of MCPyV DNA
2
Tracking Larval Behavior in zc4h2 Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For observation of free swimming, 5 dpf wild-type sibling and zc4h2 mutant larvae were placed on slide glass in embryo media. Larvae were monitored using a stereomicroscope (LEICA, MZ16), camera (LEICA DC300FX) and microscope imaging software (LEICA, IM50). For recording of free swimming, monitor display was recorded by using Camtasia Studio software (TechSmith, Vesion 7.0.0). For observation of eye, pectoral fin and jaw movement, 5 dpf larvae were transferred onto a glass slide coated with 3% methylcellulose.
3
MCPyV DNA Detection by FISH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MCPyV FISH was performed as previously described.22 , 23 In brief, deparaffinized 3 μm thick sections were pretreated with 0.2 M hydrochloric acid, incubated with 1 M NaSCN and digested with 1 mg/mL pepsin (2500–3500 U/mg, Sigma Chemical, St. Louis, MO, USA). The biotin labeled “specific” MCPyV DNA probe was added to the samples at a concentration of 5 ng/μL, followed by denaturation of DNA (five minutes, 80°C) and hybridization overnight (37°C, humid chamber; ThermoBrite System, Abbott Molecular, Abbot Park, IL, USA). Unbound MCPyV DNA probe was stringently washed away. Bound probe was detected by sequential incubation in a combination of secondary antibodies: fluorescein isothiocyanate (FITC) avidin secondary antibody (1:500) and biotin conjugated goat anti‐avidin (1:100; Vector, Brunschwig Chemie, Amsterdam, The Netherlands). Prior to incubation, aspecific binding sites were blocked with Boehringer Blocking reagent (Roche,
Molecular Diagnostics Inc., South Branchburg, NJ, USA). Cell nuclei were counterstained, and cover slipped with 4,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI; 0.2 μg/mL; Vectashield, Vector Laboratories, Burlingame, CA, USA). Samples were visualized using a DM 5000B fluorescence microscope (Leica, Wetzlar, Germany) coupled to an online digital camera (Leica DC 300 Fx) for independent evaluation of FISH signals by two investigators.
Molecular Diagnostics Inc., South Branchburg, NJ, USA). Cell nuclei were counterstained, and cover slipped with 4,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI; 0.2 μg/mL; Vectashield, Vector Laboratories, Burlingame, CA, USA). Samples were visualized using a DM 5000B fluorescence microscope (Leica, Wetzlar, Germany) coupled to an online digital camera (Leica DC 300 Fx) for independent evaluation of FISH signals by two investigators.
4
Zebrafish Hepatotoxicity and Neurotoxicity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tamoxifen (T5648, Sigma-Aldrich) or acetaminophen (A7085, Sigma-Aldrich [19 (link)]) was dissolved in DMSO. For the hepatotoxicity assay, 4 dpf (day after fertilization) zebrafish larvae were arrayed in 24-well plates (five individuals per well) containing 1 mL embryonic medium that was diluted from 1000x stock solutions of NaCl (29.4 g/100 mL, Sigma-Aldrich), KCl (1.27 g/100 mL, Sigma-Aldrich), CaCl2·2H2O (4.85 g/100 mL, Sigma-Aldrich), and MgSO4·7H2O (8.13 g/100 mL, Sigma-Aldrich), as these solutions can be autoclaved and stored at room temperature. The larvae were exposed to various doses of liver toxicants for 24 hours. For imaging, larvae were anesthetized with tricaine (MS-220, Sigma-Aldrich) and mounted on 3% methyl cellulose (Sigma-Aldrich). Mounted larvae were imaged with a Leica MZ APO stereomicroscope and DC300 FX (Leica, Japan). In addition, 3-month-old zebrafish were arrayed in 200 mL cages (3 individuals per cage) and exposed to liver toxicants for 24 hours. To induce nervous system-specific toxicity, we used the nitroreductase/metronidazole system in combination with the neuron-specific transgenic line. Nitroreductase converts the nontoxic prodrug, metronidazole (Mtz), into cytotoxic agents [20 (link)].
5
VP Lobe Histomorphometric Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At PND 540, samples of VP lobes from the CTR and GLLP groups (n = 12/group) were fixed in Methacarn [78 (link)], diaphanized in xylene, and embedded in Paraplast (Sigma, St. Louis, MO, USA). For the general morphological study, histological slices (5 µm) were stained with Hematoxylin/Eosin. The slides were evaluated using the image analyzer Leica Q-win software and a Leica DMLB 80 microscope connected to a Leica DC300FX (Version 3 for Windows).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!