Cells were visualized with infrared-differential interference contrast optics (Axioskop2; Zeiss). We performed patch-clamp recordings in the whole cell configuration at 35–37°C using the Digidata 1344A interface, the Multiclamp 700A amplifier and PClamp8 software (Axon Instruments, Foster City, CA, USA). For current-clamp recordings, patch electrodes (6–10 MΩ) contained (in mM): 120 K-gluconate, 20 KCl, 10 HEPES, 2 MgATP and 0.5 NaGTP, pH 7.2–7.4 (285–295 mOsm). Biocytin (Sigma-Aldrich; 5 mg/ml) was added in the pipette solution and osmolarity corrected when necessary. To reveal the Biocytin injected during whole-cell recordings of pyramidal neurons or interneurons, the recorded sections were fixed in paraformaldehyde (Antigenfix, 4%) for 48 h. They were rinsed in PBS and incubated for 1 h at RT in PBS/Triton 0.3%/goat normal serum 5%. They were incubated overnight at 4°C with PBS/Triton 0.3%/goat normal serum 5% and streptavidin Cy3 (1:1000). They were rinsed in PBS 10 min 5 times, mounted in Eukitt (Electron Microscopy Sciences), and coverslipped. Dendritic fields were reconstructed using the Neurolucida system (MicroBrightField Inc., Colchester, VT, USA).
Eukitt
Manufactured by Electron Microscopy Sciences
Sourced in United States
Eukitt is a rapid-drying mounting medium designed for the preparation of permanent microscope slides. It is a clear, colorless, and viscous solution that is used to mount specimens on glass slides and cover slips, providing a durable and transparent seal. Eukitt is compatible with a wide range of biological and chemical samples and is commonly used in microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488, by Thermo Fisher Scientific (2 mentions)
Abc amplification, by Vector Laboratories (2 mentions)
Alexa 564, by Thermo Fisher Scientific (2 mentions)
Sc 8430, by Santa Cruz Biotechnology (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Biocytin, by Merck Group (1 mentions)
Axioskop 2, by Zeiss (1 mentions)
Multiclamp 700a amplifier, by Molecular Devices (1 mentions)
Pclamp8, by Molecular Devices (1 mentions)
Nuclear fast red counterstain, by Vector Laboratories (1 mentions)
Nbt bcip substrate, by Roche (1 mentions)
Alkaline phosphatase conjugated anti fam, by Roche (1 mentions)
Freedom evo, by Tecan (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
5 protocols using eukitt
1
Visualizing and Characterizing Neuronal Cells
2
Immunolabeling of Mouse Brain Sections
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Mouse brain sections were prepared for immunolabeling as previously described (Crittenden et al., 2021 (link); Niemz et al., 2017 (link)). The sections were incubated with rabbit polyclonal CDGI antiserum (Crittenden et al., 2004 (link)) for approximately 12 h at 4 °C and processed either for immunohistochemistry with ABC amplification (Vector Laboratories) and DAB detection with nickel enhancement or, in conjunction with mouse anti-CalDAG-GEFII antiserum (SC-8430, Santa Cruz Biotechnology), for immunofluorescence with secondary antibodies coupled to ALEXA 564 and ALEXA 488 (Invitrogen). Fluorescent labeling of neurons expressing D1 and D2 DA receptors was detected with mCherry or EGFP filter set in sections from Drd1a-tdTomato and Drd2-EGFP BAC transgenic mice (Gong et al., 2003 (link)). Sections were mounted and coverslipped with Eukitt (Electron Microscopy Sciences) following immunohistochemistry, or with Vectashield mounting medium (Vector Laboratories) for confocal detection of genetic fluorescent labeling. The sections were viewed with Olympus BX61 and SZX7 microscopes fitted with an Olympus DP70 camera.
3
In Situ Hybridization of miR-21 in MF Skin
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Six μm thick tissue sections from paraffin embedded lesional MF skin biopsies were used for in situ hybridization as described elsewhere [50 (link), 73 (link)]. In brief, the slides were deparaffinized and placed in a Tecan Freedom Evo automated hybridization instrument (Tecan, Männedorf, Switzerland), which was programmed to perform the following steps: proteinase-K treatment using 15 μg/mL for 8 min at 37°C, pre-hybridization in formamide-free hybridization buffer (Exiqon, Vedbæk, Denmark) at 57°C for 15 min, in situ hybridization with double-FAM-labeled miR-21 and scramble LNA probes (both at 40nM, Exiqon) at 57°C for 60 min, stringent washes with SSC buffers at 57°C over 33 min followed by incubation of alkaline phosphatase-conjugated anti-FAM, NBT-BCIP substrate (all from Roche, Mannheim, Germany), and finally nuclear fast red counterstain (Vector Laboratories, Burlingname, CA, USA). Finally, all slides were dehydrated and the sections mounted with Eukitt (Electron Microscopy Sciences, Hatfield, PA, USA).
4
Visualization of Fibroblast-Titanium Nanoparticle Interaction
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Prior to exposure, the fibroblasts were seeded in two-well glass chambers (Thermo Fisher Scientific; Nunc™ Lab-Tek™) and kept for 48 hours at 37°C till they became 70%–80% confluent. They were then exposed for 24 hours to 0.5 mg/L of TiO2 NPs by removing the supernatant, washing with PBS, and replacing it with 1 mL of TiO2 NP solutions prepared as described above. At the end of the exposure, cells were washed three times with PBS in order to remove unattached particles, followed by fixation in 4% formaldehyde for 15 minutes at room temperature. The fibroblasts were then washed twice with PBS and once with sterile water. After removing the chambers from the slides, a mounting medium (Eukitt™; Electron Microscopy Sciences, Hatfield, PA, USA) was used to mount a cover-slip. Each experiment was repeated at least three times and run in duplicate. Imaging and image analysis was performed by using an ultra-high resolution dark field condenser (URI [ultra-high resolution imaging] CytoViva™ 130; Warner Instruments, Hamden, CT, USA), on an optical microscope (Olympus BX41 microscope; Olympus Corporation, Tokyo, Japan), with a light source (X-Cite™ 120; EXFO, VANIER, QC, Canada).31 (link) The fixed cells were examined with a 100× oil immersion objective.
5
Immunolabeling of Mouse Brain Sections
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