A Leica bright-field microscope (Leica Aristoplan, with digital camera using Leica 1000 software) was used to assess and acquire images from H&E-stained and peroxidase-stained sections. Immunofluorescent stains were analyzed and images acquired and processed using a Leica TCS SP2 system, a Leica TSC SP5 system, and UV CLSM acquisition software and a Leica DM600B UV microscope with FW4000 acquisition software. In the CLSM experiments, the red and green channels were imaged sequentially.
Aristoplan
Manufactured by Leica
Sourced in Germany
The Aristoplan is a high-quality optical instrument designed for industrial and scientific applications. It features a sturdy construction and precision optics to provide accurate measurements and observations.
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5 protocols using aristoplan
1
Microscopic Imaging of Stained Tissue
2
Immunohistochemical Staining of NGFR p75
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Formalin-fixed and paraffin-embedded tissue blocks were sectioned at 4 μm and mounted onto poly-L-lysine-coated slides. Immunohistochemical incubation was carried out in a BenchMark IHC Full System immunostainer (Roche Diagnostics, Mannheim, Germany) using the avidin-biotin peroxidase method with diaminobenzidine as chromogen according to the manufacturer's instructions (UltraView DAB Detection Kit, Firma Roche Diagnostics, Mannheim, Germany). Anti-NGFR p75 (rabbit polyclonal, Sigma, Germany) was used as primary antibody at a dilution of 1:400. Anonymized sections were independently evaluated semiquantitatively in a light microscope by a neuroanatomist (WN) (Leica Aristoplan, Leica, Bensheim, Germany) and a pediatric urologist (MP) (Zeiss Lab A1, Zeiss, Jena, Germany) who were both blinded to the patients' history. Density of NGFR p75 was assessed from absent, sparse, moderate dense, to dense.
3
Arthrocentesis and Crystal Examination
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Following the measurements in the dark field scanner, adapted arthrocentesis was performed. In order to obtain as much sample material as possible the joints were opened with a section knife. Imprints were made from the opened joints and the surrounding soft tissue and examined under the microscope (Aristoplan, Leica, Germany) by a veterinarian for the presence of needle-shaped crystals. Images were taken with a camera (Leica MC 190 HD, Leica, Germany) and edited with the Leica Application Suite program.
4
Stomatal Density Measurement Protocol
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In experiments 3 and 4, on 20 DAT, one stomatal imprint per biological replicate (with three biological replicates per genotype) was taken on the leaf abaxial side using the silicon rubber impression technique [53 (link)]. Stomatal density was measured on images (800-fold magnification) of the imprints (Figure S1 ) using a microscope (Leica, Aristoplan, Wetzlar, Germany) connected to a digital camera (DXM-1200, Nikon, Tokyo, Japan). Per imprint, stomata were counted on a randomly selected area of 0.25 mm2. Image processing and count of stomata was done using ImageJ (University of Texas Health Science Centre at San Antonio, TX, USA).
5
Histological Analysis of Pulp Alterations
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The samples were impregnated in low melting temperature paraffin and transverse histological semi serial sections (40 µm of interval between adjacent sections) between the cervical and middle portions of the upper right first molar for each animal were analyzed under brightfield microscope (Leica - Aristoplan, Wetzlar, Germany) coupled to an Axiocam MRc digital camera (Carl Zeiss MicroImaging GmbH, Germany).
For the histological analysis of pulp changes, the buccal root pulp of the upper right molar was evaluated by concerning the presence or absence of the following phenomena: pulp hyalinization, pulp nodules, diffuse calcification, vascular congestion, bleeding, thrombosis and dentinal tubules filled with nuclei (Massaro et al., 2009 (link)). In addition, four different histomorphological events were qualitatively analyzed (Fiane et al., 2014 (link), Nishioka et al., 1998 (link)). (a) the odontoblast layer; (b) deposition of reactionary dentin; (c) pulp necrosis; and (d) inflammatory infiltrate. All of these were defined as: absent; present in a restricted area; partially present; and totally present (Nishioka et al., 1998 (link)). To avoid bias, the examiner went through a calibration process and was unaware of which group the images belonged to (Supplementary Material 3 ).
For the histological analysis of pulp changes, the buccal root pulp of the upper right molar was evaluated by concerning the presence or absence of the following phenomena: pulp hyalinization, pulp nodules, diffuse calcification, vascular congestion, bleeding, thrombosis and dentinal tubules filled with nuclei (Massaro et al., 2009 (link)). In addition, four different histomorphological events were qualitatively analyzed (Fiane et al., 2014 (link), Nishioka et al., 1998 (link)). (a) the odontoblast layer; (b) deposition of reactionary dentin; (c) pulp necrosis; and (d) inflammatory infiltrate. All of these were defined as: absent; present in a restricted area; partially present; and totally present (Nishioka et al., 1998 (link)). To avoid bias, the examiner went through a calibration process and was unaware of which group the images belonged to (
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