For the histological study, we used different animals than those used for behavioral testing. At 14 days after cannula implantation surgery and infusion of BoNT/A-HC, animals were re-anesthetized and transcardially perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). L4 DRGs were resected, postfixed in the same fixative for 24 h, and then immersed in 20% sucrose in 0.02 M phosphate-buffered saline (pH 7.4) for 48 h. The DRGs were embedded in Tissue-Tek (Agar Scientific, Stansted, UK) optimal cutting temperature compound and flash frozen in liquid nitrogen. Frozen sections, 10 µm thick, were cut on a cryostat and mounted onto silane-coated slides (Matsunami, Osaka, Japan).
Tissue tek
Manufactured by Agar Scientific
Sourced in United Kingdom
Tissue-Tek is a line of laboratory equipment used for the processing, embedding, and staining of tissue samples. It provides an automated and standardized approach to tissue preparation for microscopic analysis.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using tissue tek
1
Histological Analysis of BoNT/A-HC Effects
2
Cryo-SEM Imaging of Leaf Samples
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Small leaf samples of about 5 mm × 5 mm were excised from freshly harvested leaves and immediately fixed on a plane cryo transfer shuttle with conductive mounting medium (1:1 mix of Tissue-Tek and colloidal graphite, Agar Scientific Ltd., Stansted, United Kingdom) and then processed according to [65 (link)]. In brief, after shock-freezing at -210 °C in nitrogen slush, the samples were transferred to a pre-cooled (-135 °C) cryo chamber (PP2000 T, Quorum Technologies Ltd., Laughton, United Kingdom) and sublimated for 15 min at -90 °C for 15 min. After sputtering with platinum (30 s coating at 5–10 mA in an Argon atmosphere), the specimens were transferred to the cryo-stage in the SEM chamber (T = -135 °C) and imaged (Quanta 250 FEG field emission scanning electron microscope, FEI, Brno, Czech Republic) under ultravacuum (3 10–7 mbar). Backscattered electrons were collected by an Everhart–Thornley detector at a working distance of 5 mm, and an accelerating voltage of 10 kV.
3
Cryogenic Scanning Electron Microscopy of Streptomyces
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Streptomyces samples were mounted on an aluminum stub using Tissue Tek (Agar Scientific, Ltd., Stansted, England) and plunged into liquid nitrogen slush. The sample was transferred onto the cryo-stage of an ALTO 2500 cryo-system (Gatan, Oxford, England) attached to an FEI Nova NanoSEM 450 field emission scanning electron microscope (SEM) (Thermo Fisher Scientific, Eindhoven, The Netherlands). Sublimation of surface frost was performed at −95°C for 4 min before sputter coating with platinum for 150 s at 10 mA. The sample was moved onto the main cryo-stage in the microscope, held at −125°C, and imaged at 3 kV, spot 3, and digital TIFF files were stored.
4
Monitoring Disease Progression in Mouse Model
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Disease progression was followed using Topical Endoscope Fundal Imaging (TEFI) at day 15, day 21 and day 24 post peptide immunization (pi) to compare S100B KO and C57BL/6 WT mice. Mice were anaesthetised, pupils dilated using 1% (w/v) tropicamide and 2.5% (w/v) phenylephrine hydrochloride (Bausch & Lomb Minims, Chauvin Pharmaceuticals Ltd, London, UK), and Viscotear liquid gel (Novartis Pharmaceuticals, Frimley, UK) was applied to each eye to provide good endoscope contact to cornea and avoid eyes drying. Fundus images obtained were clinically graded on a scale 0–4 by scoring changes in retinal infiltrate and lesions, retinal vessels and clarity of the optic disc according to Xu et al.,2008 [26 (link)]. At approximately peak disease, day 24 pi, mice were culled and eyes were snap frozen in OCT compound (Tissue-Tek, Agar Scientific, Essex, UK). Eyes were serially sectioned at 6 μm and sections fixed in acetone, haematoxylin stained and dehydrated in ethanol. Sections were used to grade disease based on cell infiltration and retinal structure, as described in Copland et al., 2008 [27 (link)]. Sections were masked and graded using 3 sections at each of 3 spaced intervals through the eye.
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