After the μTBS test, the debonded specimens were placed in epoxy resin (EpoxiCure 2, Buehler). The resin-embedded specimens were polished with wet silicon carbide papers (600, 800, 1000, 1200, 1500, and 2000-grit) and diamond pastes (6, 3, 1, and 0.25 μm) sequentially. After finishing, the specimens were cleaned ultrasonically for 3 min in distilled water. Then, the specimens were dried for 24 h, gold sputter-coated, and examined with a scanning electron microscope (SEM; JSM-5310LV, JEOL, Tokyo, Japan) at 50× and 500× magnification. The failure modes were classified into the following six categories as illustrated in Fig. 1: Type A: cohesive failure in the resin block; Type B: complete adhesive failure at the interface between block and resin cement; Type C: cohesive failure in resin cement; Type D: complete adhesive failure at the interface between resin cement and dentin; Type E: cohesive failure in dentin; Type F: mixed failure.
Epoxicure 2
Manufactured by Buehler
Sourced in United Kingdom, United States
EpoxiCure 2 is a two-component, room temperature curing epoxy resin system designed for general purpose embedding and casting applications. It offers a low viscosity and controlled cure time to facilitate use in a variety of laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Jsm 5310lv, by JEOL (2 mentions)
Jsm it100, by JEOL (2 mentions)
Elyra s 1, by Zeiss (1 mentions)
Plan neofluar 10x 0.30 m27 objective, by Zeiss (1 mentions)
Zen black software, by Zeiss (1 mentions)
Plan apochromat 63x 1.4 oil dic m27 objective, by Zeiss (1 mentions)
Dp paste p, by Struers (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Axio zoom v16, by Zeiss (1 mentions)
Bx 41 biological microscope, by Olympus (1 mentions)
Xl 3000, by 3M (1 mentions)
Isomet low speed precision cutter, by Buehler (1 mentions)
Cimarec, by Thermo Fisher Scientific (1 mentions)
Viscostat, by Ultradent (1 mentions)
Isomet, by Buehler (1 mentions)
Viscostat clear, by Ultradent (1 mentions)
Clearfil ap x, by Kuraray (1 mentions)
Clearfil se bond, by Kuraray (1 mentions)
11 protocols using epoxicure 2
1
Scanning Electron Microscopy Analysis of Adhesive Failure Modes
2
Keratin Extraction from Merino Wool
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Undyed natural unprocessed Merino wool was supplied by 80 skeins online yarn shop in Rugby, United Kingdom, with filament diameter 30 ± 5 µm, yarn diameter 0.62 mm, and filament count 440. This type of wool was used as a source of keratin protein and was kept at room temperature in a dry place. The solvent 1-ethyl-3 methylimidazolium acetate [C2mim][OAc] (purity ≥ 98%) was purchased from Proionic GmbH, Grambach, Austria. Throughout the experiments, the water content of [C2mim][OAc] was <0.2%, measured by a Karl Fischer titration machine (899 Coulometer, Metrohm U.K. Ltd., Runcorn, UK). To allow a clear image of the wool yarn cross section, a cold-curing epoxy resin (EpoxiCure 2, Buehler, Coventry, UK) was used.
3
Authi genic Carbonate FISH Analysis
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An authigenic carbonate sample from Jaco Scar (Costa Rica margin) was incubated for 1 y under the conditions described above for the sediment-free enrichment cultures. A fragment of this carbonate was then fixed in 1% PFA overnight at 4 °C, washed with 1X PBS buffer, and embedded in Epoxicure 2 (Buehler Ltd.). The resinembedded section was subsequently glued to a 1-inch round wafer, sectioned by rock saw, and polished with alumina polishing film of grits 60, 30, 12, 5, and 2 mm to achieve a rock section thickness of about 50 mm. For FISH, a dehydration series (50%, 75%, and 100% ethanol, 1 min each) was performed on the thin section, followed by hybridization with probes targeting ANME-2 [ANME-2-712 (52) and SRB [DSS658 (1) in 40% formamide hybridization buffer, as previously described (87). 5 µg/mL of a DAPI-Citifluor mounting medium was added prior to epifluorescence microscopy imaging. Epifluorescence pictures were taken with a fluorescence microscope (Elyra S.1, Zeiss) at 10x (EC Plan-Neofluar 10x/0.30 M27 objective) and 63x magnification (Plan-Apochromat 63x/1.4 Oil DIC M27 objective). Images were acquired using the Zen black software (Zeiss) and the image compositing was performed using the software Fiji (91) .
4
SEM Analysis of Fractured Dental Specimens
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After the MTBS testing, the fractured specimens were placed horizontally in epoxy resin (EpoxiCure 2, Buehler). After the resin was cured for 24 h, the resin-embedded specimens were polished with a sequence of wet silicon carbide papers (600-, 800-, 1000-, 1200-, 1500-, and 2000grit) and diamond pastes (6, 3, 1, and 0.25 μm). After finishing, the specimens were ultrasonically cleaned for 3 min in distilled water. Then, the specimens were kept in a desiccator for 24 h, sputtered with a gold coating and examined via scanning electron microscopy (SEM) (JSM-5310LV, JEOL, Tokyo, Japan) at 50× and 500× magnification. The failure modes were categorized into the following seven groups: cohesive block: cohesive failure in the CAD/CAM resin block; adhesive block/cement: complete adhesive failure at the interface between the CAD/CAM resin block and the resin cement; cohesive cement: cohesive failure in the resin cement;
adhesive cement/coating: complete adhesive failure at the interface between the resin cement and the adhesive or the resin-coating layer; cohesive coating: cohesive failure in the adhesive or resin-coating layer; interfacial dentin: complete adhesive failure at the interface between the adhesive, the resin-coating layer, or the resin cement and dentin; and cohesive dentin: cohesive failure in dentin.
adhesive cement/coating: complete adhesive failure at the interface between the resin cement and the adhesive or the resin-coating layer; cohesive coating: cohesive failure in the adhesive or resin-coating layer; interfacial dentin: complete adhesive failure at the interface between the adhesive, the resin-coating layer, or the resin cement and dentin; and cohesive dentin: cohesive failure in dentin.
5
Resin Thickness Measurement of Bonded Specimens
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Three specimens for each group were prepared in the same manner as for the µTBS test and stored in water at 37°C for 24 h. Bonded specimens were embedded in selfcuring epoxy resin (Epoxicure 2, Buehler) and sectioned into two halves using a low-speed diamond saw (Isomet). Finishing was done using a sequence of 600-, 800-, 1000-, 1200-, 1500-, and 2000-grit SiC papers under running water followed by diamond pastes down to 0.25 µm (DP-Paste P, Struers A/S, Ballerup, Denmark). In each step, specimens were cleaned ultrasonically. The specimens were kept in a desiccator for 24 h and then observed under a SEM (JSM-IT100, JEOL) at a 1,000× magnification. The thickness of adhesive and resincoating layer was analyzed using ImageJ software (Version 1.52, National instate of health, Bethesda, Maryland, USA) at 15 different points for each sample and the average was used as sample mean.
6
Sample Preparation for SEM Analysis
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Roots and occlusal surfaces of the samples were cut, then sectioned using a 0.1 mm-thick disk, across the filling, then placed in resin moldings EpoxiCure™ 2 (Buehler; Lake Bluff, IL, USA). The sample surface was polished using 600, 1000 and 1500-grit sandpapers under water cooling. Samples were sonically cleaned, the surfaces conditioned for 10 seconds using 36% phosphoric acid, then rinsed under tap water for 1 minute and immersed in 5% sodium hypochlorite for 2 minutes [22 ].
The dehydration process was subsequently performed by dipping samples into an increasingly higher concentration of ethanol, from 25% to 50%, 75%, 95% and 100% for 20 minutes at each stage. The process was repeated 3 times at the 100% concentration stage and samples were allowed to dry at room temperature for 10 minutes [23 (link)]. Prior to SEM analysis, the surface of each sample was coated with gold in a vacuum.
The dehydration process was subsequently performed by dipping samples into an increasingly higher concentration of ethanol, from 25% to 50%, 75%, 95% and 100% for 20 minutes at each stage. The process was repeated 3 times at the 100% concentration stage and samples were allowed to dry at room temperature for 10 minutes [23 (link)]. Prior to SEM analysis, the surface of each sample was coated with gold in a vacuum.
7
Scanning Electron Microscopy Analysis of Fractured Dental Specimens
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After the MTBS test, the fractured specimens were placed horizontally in epoxy resin (EpoxiCure 2, Buehler). The resin-embedded specimens were polished after 24 h with a sequence of wet silicon carbide papers (600-, 800-, 1000-, 1200-, 1500-, and 2000-grit) and diamond pastes (6, 3, 1, and 0.25 μm). The specimens were ultrasonically cleaned for 3 min in distilled water. Then, the specimens were kept in a desiccator for 24 h, sputtered with a gold coating and examined by scanning electron microscopy (SEM; JSM-IT100, JEOL, Tokyo, Japan) at 50× magnification. The failure modes were classified into the following seven categories, as illustrated in Fig. 1: blo: cohesive failure in the resin block; blo-cem: complete adhesive failure at the interface between resin block and resin cement; cem: cohesive failure in resin cement; cem-cr: complete adhesive failure at the interface between resin cement and resin-coated dentin; cr: cohesive failure in resin-coating material; inter-den: complete adhesive failure between resin-coating material and dentin; and den: cohesive failure in dentin. When pre-test failures occurred during specimen preparation for MTBS, failure mode analysis was carried out.
8
Imaging Microbial Mat and Sinter Samples
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Free-living surface microbial mat samples were imaged directly with an Olympus BX-41 biological microscope. Hard sinter samples were prepared for both thin (100 μm) and thick (∼5 mm) sections. These samples were first air-dried in a sterile laminar fume hood for 3–7 days while monitoring weight loss. After drying, a subset of samples was mounted in epoxy resin (EpoxiCure 2; Buehler) under three to five vacuum cycles to remove air bubbles.
These samples were further cut and polished with a diamond blade lubricated with pure mineral oil (CAS: 8042-47-5, Sigma-Aldrich: 415080010) to prevent dissolution of salts. Selected sections were scanned with a Zeiss Axio Zoom.V16 motorized stereo microscope (IBPS, UPMC) by using combined transmitted and reflected light to obtain large area images at 1000 × magnification (corresponding to ∼1 μm/pixel). Higher resolution optical images in selected areas were also obtained with the Olympus BX-41 microscope.
These samples were further cut and polished with a diamond blade lubricated with pure mineral oil (CAS: 8042-47-5, Sigma-Aldrich: 415080010) to prevent dissolution of salts. Selected sections were scanned with a Zeiss Axio Zoom.V16 motorized stereo microscope (IBPS, UPMC) by using combined transmitted and reflected light to obtain large area images at 1000 × magnification (corresponding to ∼1 μm/pixel). Higher resolution optical images in selected areas were also obtained with the Olympus BX-41 microscope.
9
Dentin Disk Sandwich Preparation for ABRZ
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Figure 2 illustrates the specimen preparation for the ABRZ observations. For each group, three molars were sectioned into 2-mm-thick dentin disks at the midcoronal portion horizontal to the tooth axis. Each primer was applied on the dentin surface for 20 s and bonding agent was applied and light-cured (XL 3000, 3M) for 10 s. Then, Estelite flow quick (Shade A2, Tokuyama Dental, Tokyo, Japan) was placed between pairs of treated dentin disks and light-cured for 40 s from the top and bottom surfaces to form a dentin disk sandwich 3) . After 24-h storage or TC5000 treatment, samples for ABRZ observation were embedded in epoxy resin (Epoxicure 2, Buehler). Then, the acid-base challenge was performed
10
Preparation of Undecalcified Bone Sections
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Standard histological methods were followed to prepare thin sections (Bancroft & Gamble, 2008 ). Using a portable hotplate (Thermo Fisher Scientific Cimarec®), extracted bone sections were gently macerated in a warm water and enzymatic laundry detergent mix over low heat (55 ± 5°C) for approximately 12 h, with the water and detergent mix replenished three times (Uhre et al., 2015 (link)). This is a quick, effective, and gentle process that does not damage the bone microstructure. Adhering soft tissue was then removed with the assistance of a plastic spatula and soft toothbrush. The undecalcified bone samples were placed in a fume cabinet to air dry for 24 h before being dehydrated in a graded ethanol series and cleared with xylene (An et al., 2003 ). Each section was embedded in resin (Buehler EpoxiCure™2). Sections of ~400 μm thickness were cut from the embedded blocks using a Buehler® IsoMet™ Low Speed Precision Cutter with a diamond blade, to obtain a transverse cross‐section (Bancroft & Gamble, 2008 ). Each section was ground using a Gemmasta™ GF4 faceting machine with a diamond lap, and then hand‐polished to 100 ± 25 μm thickness using diamond paste and a polishing cloth, before being washed, dried, cleaned in an ultrasonic bath, dehydrated, and mounted onto a glass microscope slide and sealed with a cover slip (Bancroft & Gamble, 2008 ).
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