Mussel shells were cleaned, dried (60 °C for 48 hours) and embedded in epoxy resin (EpoxyCure, Buehler) blocks. Embedded shells were sliced transversely using a diamond trim saw blade to section the whole length of the shell. New growth was determined through calcein staining of growth bands at the start of experimental culture as detailed in8 (link)22 ; any growth prior to this stained growth band was named old growth which occurred prior to experimental culture. The new growth at the outer edge of the shell (containing newest calcite) and towards the newest aragonite formation (containing both newest aragonite and older calcite) was sectioned, and mounted in a resin block before polishing the cut edge of the shell. Resin blocks were ultra-polished using aluminium oxide (0.3 and 1 μm) and colloidal silica (0.6 μm). The sections of ~2 mm were then prepared as thin sections into discs and polished through to 0.06 mm colloidal silica.
Epoxycure
Manufactured by Buehler
Sourced in United States
EpoxyCure is a two-component epoxy resin system designed for general purpose bonding, sealing, and coating applications. It is a low viscosity, room-temperature curing epoxy that provides high strength and excellent adhesion to a variety of substrates.
Automatically generated - may contain errors
6 protocols using epoxycure
1
Mussel Shell Growth Characterization
2
Nanoindentation Analysis of Demineralized Dentin
3
Mussel Shell Microstructure Analysis
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Mussels sampled after 9 months of experimental culture were dissected and shells cleaned and oven‐dried by incubation at 60°C for 48 h and then embedded in epoxy resin (EpoxyCure, Buehler) blocks. Embedded shells were sliced transversely using a diamond trim saw blade to section the whole length of the shell. New growth was determined through calcein staining of growth bands at the start of experimental culture as detailed by Fitzer et al. (2014b ); any growth prior to this stained growth band is termed old growth which occurred prior to experimental culture. Resin blocks were ultra‐polished using aluminum oxide (0.3 and 1 μm) and colloidal silica (0.6 μm). Shell aragonite and calcite thickness were determined using light microscopy, and aragonite/calcite ratios were calculated for comparison between populations of mussels (n = 4) in each experimental condition. General linear model (GLM) ANOVAs (Minitab 17.1.0) were used to assess the significance of the effect of pCO2 and increased temperature on aragonite and calcite thickness and aragonite/calcite ratio with assumptions of normality and homogeneity of variance being met. Graphical methods (residual plots) were used to confirm that the data fitted a normal distribution using a frequency residual histogram and normal probability plot in Mintab 17.1.0 for the GLM ANOVA.
4
Resin-Dentin Interface Preparation and Imaging
5
Fluorescent Microscopy of Mussel Shell Growth
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Shell growth and thickness of aragonite deposited since the calcein staining at the start of the experimental period, was measured using fluorescent microscopy (Olympus BH-2). Mussel shells were dried by incubation at 60°C for 48 hours, and then embedded in epoxy resin (EpoxyCure, Buehler) blocks. Embedded shells were sliced transversely using a diamond trim saw blade to section the whole length of the shell. Resin blocks were then polished. Calcein was detected under blue light excitation at 488 nm, where calcein fluoresces yellow-green27 28 . Mussel shell growth (mm) was measured using microscope image analysis software (Microtec IS500 5MP camera software) as distance from stained calcein line to the tip of the new growth. Calcite and aragonite thickness was also measured (mm) using the microscope image analysis.
6
Failure Analysis of Dental Impressions
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In case of any failure an impression (Ultra Light and Heavy body Aquasil, Dentsply, Mildford, USA) was made from the failure site after cleansing the surface with absorbent paper and hypochlorite 0.5%. Impressions were poured with cold mounting epoxy resin (EpoxyCure, Buehler, IL, USA) which was subsequently sputter-coated with a 3 nm thick layer of gold (80%) / palladium (20%) (90 s, 45mA; Balzers SCD 030, Balzers, Liechtenstein) and analysed using cold field emission Scanning Electron Microscopy (SEM) (LEO 440, Electron Microscopy Ltd, Cambridge, United Kingdom).
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