Coverslip with thickness of 0.13 mm, circular diameter of 18 mm (Marienfeld Superior, Lauda-Königshofen, Germany) were cleaned with 70% ethanol and then placed on Cimarec + ™ Hotplate (Thermofisher Scientific, MA, United States) pre-set at 120°C. 100 μL of 2.0–2.4 μm polystyrene beads (Spherotech, Lake Forest, United States) in 50% ethanol were loaded onto each Coverslip. Coverslips were heated at 120°C for 10 min and transferred to a storage dishes for experiment.
Cover slip
Manufactured by Marienfeld
Sourced in Germany
A cover slip is a thin, flat piece of glass or transparent plastic used in microscopy to cover and protect a specimen on a microscope slide. It serves to create a flat, even surface over the specimen, allowing for better optical clarity and focus during microscopic observation.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Mowiol, by Merck Group (2 mentions)
S9378, by Merck Group (2 mentions)
Cimarec hotplate, by Thermo Fisher Scientific (1 mentions)
Polystyrene beads, by Spherotech (1 mentions)
Dm2000 optical microscope, by Leica (1 mentions)
Trypan blue solution, by Merck Group (1 mentions)
Chlorhexidine digluconate, by Merck Group (1 mentions)
Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Facsverse flow cytometry, by BD (1 mentions)
Anti cd40, by Thermo Fisher Scientific (1 mentions)
Anti pd l1, by Thermo Fisher Scientific (1 mentions)
Anti mhcii, by Thermo Fisher Scientific (1 mentions)
Jawsii, by American Type Culture Collection (1 mentions)
Observer d1 fluorescence microscope, by Zeiss (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Lsm 880 airyscan, by Zeiss (1 mentions)
Superfrost plus, by Avantor (1 mentions)
Normal goat serum (ngs), by Bio-Rad (1 mentions)
8 protocols using cover slip
1
Coverslip Preparation with Polystyrene Beads
2
Measuring Intracellular Calcium Dynamics
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The resting [Ca2+]i and ER-dependent Ca2+ release were evaluated in untransfected and mutant clones loaded with the Ca2+-sensitive fluorophore, Fura-2/AM (Molecular Probes Europe BV, Leiden, The Netherlands). The Ca2+ imaging set-up and loading procedures were extensively described elsewhere [17 (link)]. Briefly, 2×105 cells were plated on 9 mm diameter cover slip (Marienfeld GmbH, Lauda-Königshofen, Germany) in a 6 well plate. Cells were incubated with 4 μM FURA-2/AM in 150 mM NaCl, 6 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 10 Mm glucose and 10 mM Hepes (PSS) for 20 min at 37°C in the dark. Cells were then washed in PSS solution and analysed with a fluorescence microscope equipped with Zeiss Achroplan 40X lens. Cells were excited at 340 and 380 nm and the light emitted was revealed at 510 nm. The calibration of resting intracellular Ca2+ levels was done using Grynkiewicz, as shown in [18 (link)]. UTP (100 μM) was added for 5 min, in absence of external calcium (0 Ca2+), to stimulate calcium release from ER through the IP3R channel.
3
Evaluating Oxidant Toxicity on Oral Cells
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The toxicity of the oxidant solution was evaluated using the Trypan blue method on oral epithelial cells present in saliva after desquamation. Trypan blue that enters living cells is immediately expelled: the living cells do not have a colored appearance whereas the dead cells appear colored in blue. The saliva of 6 adults (5 women and 1 man aged 25–65 years) was collected in sterile containers (Deltalab™). A volume of 50 µL of saliva diluted twice (in 0.9% NaCl) was mixed with 50 µL of a Trypan blue solution (Merck™) at a concentration of 0.4 g/100 mL. The percentage of dead cells (on a total of at least 120 cells in each preparation) was observed between a slide (Thermo Scientific™, Braunschweig, Germany) and coverslip (Marienfeld, Lauda-Königshofen, Germany) on a Leica DM2000 optical microscope (Leica™, Wetzlar, Ger-many). A 30-minute incubation (room temperature) of 100 µL of saliva with 100 µL of iodine–thiocyanate complexes solution or 0.4% (w/v in phosphate buffer) chlorhexidine digluconate (Sigma-Aldrich™) preceded the microscopic examination to check the toxicity of these compounds.
4
Immortalized DC Line Response to MSNs
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The immortalized immature DC line JAWS II was purchased from ATCC. JAWS II cells were seeded onto a cover slip (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Baden-Württemberg, Germany) and incubated with 250 μg/mL of rhodamine-preloaded MSNs for two days. Cells were fixed with 4% formaldehyde and permeabilized with 0.01% Triton X-100. Cells were stained with Alexa-Fluor488-conjugated phalloidin (Life Technologies, Eugene, OR, USA) and DAPI (Sigma-Aldrich, St. Louis, MO, USA). Fluorescence images were acquired using a Zeiss Observer D1 fluorescence microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany). The activation of JAWS II cells by CM-MSNs was determined by flow cytometry. JAWS II cells (5 × 106 cells) were seeded in 12-well plates and incubated with 100 or 200 μg/mL of CM-MSNs for two days. Cells were stained with anti-CD40 (BD562846), anti-CD80, anti-CD86, anti-MHC I (BD742859), anti-MHC II and anti-PD-L1 (12-5982-82, eBioscience, San Diego, CA, USA). Stained cells were analyzed by a BD FACSVerse flow cytometry.
5
Spinal Cord Tissue Sectioning and Immunolabeling
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Following postfixation and cryo-protection in sucrose (Sigma, S9378) the spinal cords were mounted using 40% sucrose in 1× PBS; 30-µm free-floating transversal sections were produced (Leica, SM2000R) and kept in 1× PBS with 0.1% NaN3 (Sigma, S-2002) in a 24-well plate (Nunc, 142475). Primary antibody (Table 2 ) was diluted in blocking buffer: 0.3% Triton X-100 (Sigma, 93443), 5% normal donkey serum (Sigma, S30), 5% BSA (Sigma, A2153), 1× PBS, and NaN3. Sections were incubated in primary antibody with gentle shaking (IKA, MS 3 digital) at 4°C O/N. Sections were washed three times in 1× PBS with gentle shaking and incubated in secondary antibody (Table 2 ) at 4°C O/N. Sections were washed three times in 1× PBS and then incubated in nucleic acid stain (Hoechst 33258, Invitrogen H3569) for 20 min at 4°C with gentle shaking. Slides were once again washed three times and placed on slides (VWR, Superfrost Plus, 48311-703). A small amount of Mowiol (Sigma, 81381) was added and the sections protected using a cover slip (Marienfeld, 010243). The stained sections were imaged using a confocal microscope (Zeiss, LSM 880 Airyscan).
6
Cryopreservation and Sectioning of Spinal Cords
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Post-fixed spinal cords were cryo-protected in 15% and 30% sucrose (Sigma, S9378) in 1×PBS. Spinal cords were mounted in cryomolds (Tissue-Tek® Cryomold®, 420572) using compound (Tissue-Tek® O.C.T.™ ) and rapidly frozen to − 60 °C. Twenty-micrometer coronal sections were produced using a cryostat (Leica, CM1850, − 22 °C) and mounted on slides (VWR, SuperFrost® Plus, 48311-703). Sections were thawed, rehydrated in 1×PBS, blocked for 2 h at RT in blocking solution (0.3% Triton X-100 (Sigma, 93443), 5% normal goat serum (Serotec, 301104, 1×PBS and 0.01% sodium azide (Sigma, S-2002)). Primary antibody (Table 1 ) was added, and sections were incubated at 4 °C for 24 h. Sections were rinsed in 1×PBS followed by incubation in secondary antibody (Table 1 ) at RT for 1 h. Sections were incubated at RT for 20 min with nucleic acid stain (Hoechst 33258, Invitrogen™ H3569). Prior to confocal microscopy, the slides were rinsed and mounted using Mowiol (Sigma, 81381) and a cover slip (Marienfeld, 010243).
7
Serum IgG and IgM Immunofluorescence Assay
8
Hoechst 33342 Staining of Blastocysts
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Blastocysts were incubated in 500 µL of washing medium with Hoechst 33342 (1 µg/mL) for 15 min at RT. After staining, the whole blastocysts were mounted in glycerol on a glass slide (Paul Marienfeld, Germany) and flattened under a coverslip (Paul Marienfeld). Digital photographs were obtained using an inverted microscope (Carl Zeiss, Germany) capable of UV illumination and equipped with excitation filters (460 nm for fluorescence, Fig. 3B ). The cells in the images were counted.
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