Spotlight 400 ft ir imaging system

The polysaccharides were isolated from G. lucidum aqueous extract following a protocol previously described by Kan et al. (2015) applying the following experimental conditions: ratio of water to material: 30 mL g⁻¹; extraction time and temperature: 120 min and 60 °C, respectively [42 (link)]. After the extraction was completed, cooling, centrifugal separation (6000 rpm, 10 min, 4 °C), and filtering were carried out in sequence to obtain a supernatant fluid containing G. lucidum polysaccharides (GLP). Finally, the GLPs were lyophilized for 24 h (LyoQuest-55, Telstar Technologies, Terrassa, Spain) using the condenser temperature of −52 °C and 0.100 mBar as the vacuum value. Dry GLP extract was analyzed at 4000–600 cm⁻¹ at 25 °C using a Spotlight 400 FT-IR Imaging System (PerkinElmer, Waltham, MA, USA) to determine the organic functional groups of GLPs.

Kidneys were processed for localization and identification of crystals. 10 μm kidney cuts were performed in a sagittal axis from the convexity to the pedicle as previously described^{8 (link)} and then analyzed using a polarizing microscope, an infrared imager (Spotlight 400 FT-IR imaging system from Perkin Elmer) and a Field Emission Scanning Electron Microscope (Zeiss SUPRA55-VP SEM) with no coating. Quantification of CaOx crystals was carried out by examining the sections at magnification ×100. Instead of quantification of the number of crystals per section which was difficult due to the presence of crystal aggregates, we expressed the number of sections (expressed as percentage) with at least one crystal detected (20 sections were analysed in a blinded fashion for each kidney). HLP and HLP + FGF7 groups were compared at different time points.

Four-μm-thick PP film (Ultralene, SPEX SamplePrep, NJ, USA) was used as a substrate for mounting thin bone sections prepared by a frozen sectioning procedure (Fig 1). The film was coated with commercial glue (Cryoglue type I, Leica Microsystems, Tokyo, Japan), which was used to fix the thin bone sections (Fig 1). The glue was diluted with n-hexane (glue:n-hexane = 9:1), and spread using a spin coater (MS-A100, Mikasa, Tokyo, Japan) at 2000 rpm for 10 sec at room temperature. All PP film, glue, and PP film + glue samples were mounted on BaF₂ substrates, and their FTIR spectra were collected using a Spotlight 400 FTIR Imaging System (PerkinElmer, MA, USA) with a single element Mercury Cadmium Telluride (MCT) detector in transmission mode at a spectral resolution of 4 cm^-1, aperture size of 100 μm x 100 μm, and with 32 scans in the mid-IR region from 4000 to 680 cm^-1. The background spectra were collected through the BaF₂ window. FTIR spectra underwent baseline correction, which was automatically applied across the whole spectrum (4000 to 680 cm^-1), using Spectrum 10 software (PerkinElmer, MA, USA). The spectra were normalized against 1 absorbance with respect to the highest band.