Glass slides (Carl Roth) were washed in 1 M KOH and in deionized MilliQ water for 15 min, glass cover slips (Carl Roth) were plasma cleaned in N2 plasma for 10 min (Diener Electronic). The slides and cover slips were assembled to measurement slides with ∼10 μl channels that were coated with biotinylated-BSA and streptavidin as described (25 ). The channels were flushed with 200 μl immobilization buffer with adjusted concentrations of MgCl2 and adenine, repeatedly flushed with 30 μl of a 100 pM RNA sample until a sufficient surface density of single molecules was achieved (∼0.1 molecules/μm2) and flowed with 125 μl imaging buffer (25 mM K2HPO4/KH2PO4 pH 7.0, 50 mM KCl, 9% (w/w) glucose, 14 U/ml glucose oxidase, 1000 U/ml catalase, sat. trolox and MgCl2 and adenine as specified) for measurement. All measurements were carried out at ambient temperature (22°C) with 532 nm laser excitation on an objective-type total internal reflection microscopy setup with an EMCCD camera (iXon, Andor Technology) at 100 ms integration time.
Glass slide
Manufactured by Carl Roth
Sourced in Germany
Glass slides are flat, transparent pieces of glass, commonly used in various laboratory and scientific applications. They provide a stable, flat surface for mounting and observing samples under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Axiovision 4, by Zeiss (2 mentions)
Glass coverslip, by Carl Roth (1 mentions)
Emccd camera, by Oxford Instruments (1 mentions)
Isopropyl alcohol, by Carl Roth (1 mentions)
Sledge microtome, by Leica (1 mentions)
Histosec, by Merck Group (1 mentions)
3d discovery, by RegenHU (1 mentions)
Glymo, by Merck Group (1 mentions)
Neutravidin, by Thermo Fisher Scientific (1 mentions)
Water purification system, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Imager z1, by Zeiss (1 mentions)
Xylene, by Carl Roth (1 mentions)
Microtome, by Leica (1 mentions)
Las v 4, by Leica (1 mentions)
Ethanol, by Carl Roth (1 mentions)
Aqueous ammonia solution, by Avantor (1 mentions)
Pfots, by Merck Group (1 mentions)
Tetraethyl orthosilicate, by Merck Group (1 mentions)
12 protocols using glass slide
1
Single-molecule RNA Immobilization and Imaging
2
Tissue Preparation for Histology
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The tissues were fixed with 4% formaldehyde in tris-buffered saline (TBS) and dehydrated by a graded ethyl alcohol (EtOH, Carl Roth GmbH, Karlsruhe, Germany) series increasing in concentration (30%, 50%, 70%, 80%, 90%) diluted in TBS, and finally in 100% EtOH. The EtOH was then replaced by isopropyl alcohol (Carl Roth GmbH) before they were embedded in paraffin wax (Histosec®, Merck KG, Darmstadt, Germany) and mounted. The paraffin block was cut into thin sections (4-8 µm thickness) using a sledge microtome (Leica Microsystems, Wetzlar, Germany). Each GI section (esophagus, stomach, duodenum, jejunum-ileum, ileum-colon and colon-rectum) was cut in three blocks with intermediate pieces of 400 µm in length between blocks. These separating pieces of tissue between blocks were discarded. Serial sections were prepared on glass slides (Carl Roth GmbH) precoated with 0.2% chrome alum-0.5% gelatine subbing solution (Merck KG). Afterwards, the sections were deparaffinised and rehydrated by a graded EtOH series decreasing in concentration (98%, 90%, 70%, 50%, 30%) ending in water. After drying the samples were stored dust-free until further use.
3
Fibronectin Coating for Cell Adhesion
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Glass slides (Carl Roth, cat. no.: P231.1) were incubated with 200 µl of 25 mg mL−1 fibronectin in PBS for 2 h at RT. After incubation slides were directly transferred to cell culture medium for further use.
4
Comparative 3D Printing of Alginate-Nanocellulose Bioinks
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3D printing was executed with two different pneumatic extrusionbased systems (n printer = 2) that were positioned in the same room for comparable ambient conditions (temperature 22 • C ± 1 K). The 3DDiscovery (regenHU, Villaz-St-Pierre, Switzerland; abbreviated RH) and the BioX (Cellink AB, Gothenburg, Sweden; BX) were equipped with identical consumables, in detail nozzles (Cellink) and glass slides (Carl Roth, Karlsruhe, Germany). Accordingly, the printing parameters were chosen as similar as possible (see Tables S1 andS2, Supporting Information). The model scaffold (Figure 1) was 3D printed with three different inks (n materials , ink = 3) and a polylactic acid (PLA) reference material (n materials , reference = 1).
The first ink was an alginate -nanocellulose blend that is crosslinked with an aqueous CaCl 2 -solution (Bioink, Cellink) directly after the extrusion process, in the following referred to as "alginate ink." The crosslinking procedure included the dripping of the liquid over the printed object, its immersion and the removal of residual moisture with a wiping tissue after 20 min of incubation as described by the manufacturer. The material was provided in ready-to-use cartridges (3 ml) and used as received after warming it up to room temperature for 1-2 h, yielding three objects per cartridge (n objects,cartridge = 3).
The first ink was an alginate -nanocellulose blend that is crosslinked with an aqueous CaCl 2 -solution (Bioink, Cellink) directly after the extrusion process, in the following referred to as "alginate ink." The crosslinking procedure included the dripping of the liquid over the printed object, its immersion and the removal of residual moisture with a wiping tissue after 20 min of incubation as described by the manufacturer. The material was provided in ready-to-use cartridges (3 ml) and used as received after warming it up to room temperature for 1-2 h, yielding three objects per cartridge (n objects,cartridge = 3).
5
Glass Slide Surface Functionalization for Bioassays
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Glass slides (Carl Roth, cat. no.: P231.1) were washed in methanol (MeOH), dried and immersed in a 1:3 (v:v) solution of 30% (w/w) hydrogen peroxide and 95% (w/w) sulfuric acid for 40 min at 70 °C. After washing three times in deionized water and two times in MeOH, the Glass slides were immediately immersed in a 5% (v/v) (3-glycidyloxypropyl)trimethoxysilane (GLYMO, Sigma, cat. no.: 440167) solution in MeOH for 16 h at room temperature. The Glass slides were subsequently washed three times in 2-propanol and dried at 105 °C for 1 h (after this, glass could be stored in a dry place)24 . The slides were then coated with 5 mg mL−1 NeutrAvidin (Thermo Scientific, cat. no.: 31050, dissolved in PBS) overnight at room temperature (RT), washed three times with PBS, and before final incubation in 3 mg ml−1 PhyB1–651 (in elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0)) for 2 h at RT in darkness.
6
Functionalized Surfaces for Bacterial Adhesion
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37% hydrochloric acid (HCl), sodium hydroxide (NaOH), hydroxylamine hydrochloride (NH2OH·HCl), 3-glycidyloxypropyltrimethoxysilane (GOPTS) and silver nitrate (AgNO3) were purchased from Sigma-Aldrich (Taufkirchen, Germany) and used without further purification. Diamino-PEG 2000 (2000 g mol−1) was a gift from Huntsman Holland. LB broth medium, glass slides (26 × 6 × 1 mm), and adhesive tape were purchased from Carl Roth (Karlsruhe, Germany). Ultrapure water (18.2 MΩ cm−1) produced using a Millipore water purification system was used as solvent. E. coli DSM 1116 shock frozen strains were purchased from DSM nutritional products (Grenzach, Germany). E. coli CFT 073, Ps. aeruginosa PAO1 and Pr. mirabilis PRM1, in LB medium and artificial urine were obtained from the strain collection of the Max-von-Pettenkofer-Institute.
7
Localizing GFP-tagged Proteins by Fluorescence Microscopy
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Cells expressing GFP-tagged proteins were grown to an early log phase at 30°C in YPG or selective media and then shifted to 23°C or 37°C for 2–4 h. Subsequently, cells were pelleted, washed with water, applied onto glass slides (Carl Roth), and examined by fluorescence microscopy without fixation, at room temperature, using a microscope (Imager Z1; Carl Zeiss) equipped with a 100/63×, NA 1.4 Plan Apochromat oil immersion objective lens (Carl Zeiss) and a digital interference contrast third generation or high efficiency EGFP filter set. Pictures were acquired with a camera (AxioCam MRm; Carl Zeiss) and AxioVision 4.8.2.0 software (Carl Zeiss) at a resolution of 1,388 × 1,040 (binning = 1 × 1, gain factor = 1). Pictures were exported as TIFF files and processed in Photoshop CS 6 (Adobe) for levels. The detailed procedure to localize GFP-tagged proteins using fluorescence microscopy has been described in Bassler et al. (2006) (link).
8
Histological Evaluation of Cell-Biomaterial Interactions
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The biomaterial-cell-complexes were fixed with 4% paraformaldehyde (PFA, Roth, Germany) for 20 min at 4 °C followed by two washing steps in distilled water. The cell biomaterials complexes were dehydrated in ethanol (Roth, Germany), were cleared in xylene (Roth, Germany), and were processed for paraffin embedding. Sections of 6 μm were produced using a microtome (Leica, Germany), were transferred to glass slides (Roth, Germany), and were processed for hematoxylin and eosin histological staining to get an overview of cell-biomaterial interaction. The sections were mounted using Eukitt mounting media (HICO-MIC, Hirtz & Co, Germany) and were examined using the light microscope equipped with a digital camera and the LAS V4.4 software (Leica, Germany).
9
Facile Fabrication of Superhydrophobic Surfaces
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All experiments were carried out at room temperature. The following chemicals were used: Tetraethylorthosilicate (TEOS, 98%, Sigma-Aldrich, Germany), 1H,1H,2H,2H-perfluorooctyltrichlorosilane (PFOTS, 97%, Sigma-Aldrich, Germany), trichloromethylsilane (TCMS, 99%, Sigma-Aldrich, Germany), 1H,1H,2H,2H-perfluorodecyltrichlorosilane (PFDTS, 98%, Alfa Aesar, Germany), hexane (99,99%, Fisher Chemical, Germany), aqueous ammonia solution (28 wt.%, VWR International and Normapur, Germany), and photoresist SU-8 (Microchem, Germany). Reagents were used as received. Paraffin candles were obtained from the local supermarket. Polished silicon wafers were obtained from Si-Mat (Germany). Glass slides of 24 × 60 mm 2 and 170±5 µm thickness were obtained from Carl Roth (Germany).
10
Histological Analysis of Gingival Inflammation
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Gingival biopsies from both groups were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 2 days, followed by hydration and dehydration in an ascending ethanol series (AppliChem, Darmstadt, Germany), and subsequently embedded in paraffin (McCormick Scientific, Richmond, IL, USA). Next, tissue sections of 2.5-μm thickness were sliced, mounted on glass slides (Carl Roth), and dried at 37 °C overnight. Then, all sections were stained with hematoxylin and eosin (H&E) (Merck Eurolab, Darmstadt, Germany), dehydrated, and mounted with DePeX (SERVA Electrophoresis GmbH, Heidelberg, Germany). Finally, stained tissue sections were used to analyze the presence or absence of gingival inflammation by using an Axioskop 2 microscope (Carl Zeiss, Jena, Germany) with an AxioCam MRc camera and the AxioVision 4.7 software (Carl Zeiss).
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