The VNO was removed from the two heads used for macroscopic anatomy by inserting a scalpel between the vomeronasal cartilage and the nasal septum, and then embedded in paraffin using a standard procedure. The nasal parts of the other three heads were decalcified in Plank–Rychlo solution [12 (link)] for 3 h, then embedded in paraffin. Specimens were sequentially cut into 5-μm-thick transverse sections by using an LS-113 sliding microtome (Yamato Kohki Industrial Co., Ltd., Saitama, Japan) at 20-μm intervals. The sections were deparaffinized, stained with hematoxylin-eosin, periodic acid-Schiff (PAS) or Alcian blue (pH 2.5) [13 (link)], and then assessed using a Microphot-FX microscope (Nikon, Tokyo, Japan) equipped with a Digital Sight DS-5 M camera (Nikon, Tokyo, Japan).
Digital sight ds 5m camera
Manufactured by Nikon
Sourced in Japan
The Digital Sight DS-5M is a Nikon camera designed for laboratory and industrial applications. It features a 5-megapixel CMOS sensor and supports live video capture. The camera connects via USB and is compatible with a range of Nikon lenses.
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Lab products found in correlation
Microphot fx microscope, by Nikon (2 mentions)
Nis elements f2, by Nikon (2 mentions)
Nis element image software, by Nikon (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Ds fi1 u3 camera, by Nikon (1 mentions)
Alphaphot 2 ys2 microscope, by Nikon (1 mentions)
Microphot fx, by Nikon (1 mentions)
Mgk s, by Matsunami (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Bx50 microscope, by Olympus (1 mentions)
Eclipse ts100 microscope, by Nikon (1 mentions)
Vectashield medium, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
H 1000 mounting medium, by Vector Laboratories (1 mentions)
Alexa fluor 488 goat anti mouse immunoglobulin g igg, by Thermo Fisher Scientific (1 mentions)
Ellipse 80i microscope, by Nikon (1 mentions)
Dylight 549 anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
10 protocols using digital sight ds 5m camera
1
Histology of the Vomeronasal Organ
2
Histological Processing and 3D Reconstruction
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The histological processing involved sample fixation in dilute (1:2) Bouin’s fluid, dehydration in a graded ethanol series, clearing in xylene, paraffin-embedding (Histoplast, Argentina), and sectioning (5–10 μm-thick) on a rotary microtome (Microm). After staining with Gill’s hematoxylin-eosin, we examined and photographed the samples under a Nikon Eclipse 80i microscope using Nikon DS-Fi1-U3 camera and Nikon NIS-ELEMENT Image Software.
Also, we examined serially sectioned (10 μm-thick) Harris hematoxylin-eosin-stained lung samples under a Nikon Alphaphot-2 YS2 microscope equipped with a Nikon Digital Sight DS-5M camera. We selected the images of every fifth section for the 3D-reconstruction of the blood system and innervation of the lung. For this purpose, we used the software Reconstruct 1.1.0.0 (Fiala, 2005 (link)) and followed the procedure described by Ruthensteiner & Heß (2008) (link) to assemble the final PDF-model, as described in Rodriguez et al. (2019) (link).
Also, we examined serially sectioned (10 μm-thick) Harris hematoxylin-eosin-stained lung samples under a Nikon Alphaphot-2 YS2 microscope equipped with a Nikon Digital Sight DS-5M camera. We selected the images of every fifth section for the 3D-reconstruction of the blood system and innervation of the lung. For this purpose, we used the software Reconstruct 1.1.0.0 (Fiala, 2005 (link)) and followed the procedure described by Ruthensteiner & Heß (2008) (link) to assemble the final PDF-model, as described in Rodriguez et al. (2019) (link).
3
Spatial Mapping of Turtle Oviduct
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We dissected and embedded 29 parts of the oviduct of one turtle (ID: 035) in paraffin, after fixing. The sampled area comprised four from within the infundibulum (fimbria, and distal, middle, and proximal tubular areas), 17 at equal distance in the magnum, two in the isthmus (distal and proximal areas), three in the uterus (distal, middle and proximal areas), and three areas around the border between the uterus and vagina (proximal end of the uterus, transition area, and distal end of the vagina). The oviducts of the other two turtles (ID: 024 and 028) were dissected into 12 parts, comprising four in the infundibulum, three in the magnum (distal, middle, and proximal areas), two in the isthmus, and three in the uterus. Embedded samples were sliced into 5-μm-thick sections, deparaffinized, stained with hematoxylin–eosin, and assessed using a Microphot-FX microscope equipped with a Digital Sight DS-5M camera (both from Nikon Corp., Tokyo, Japan).
4
Body Length Measurement Protocol
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To determine body lengths under the various experimental treatments, animals were fixed in 4% Paraformaldehyde (PFA)/0.1 M MOPS (pH 7.0)/0.5 M NaCl/5 mM EGTA/0.2% TritonX-100 at room temperature (RT) for 1 h. Body length was then determined using a Nikon SM1500 stereo microscopic equipped with a Nikon digital sight DS-5M camera. Measurement of body length was aided by the use Nikon Eclipse Net imaging software.
5
Histological Analysis of Spleen Tissues
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Spleen samples were fixed in 10% neutral buffered formalin. Later, tissue samples were dehydrated through a series of graded alcohol, embedded in paraffin, and sectioned at a thickness of 4 µm. After sections were deparaffinized, staining of tissue sections was performed with hematoxylin & eosin (H & E). The slides were mounted on MGK-S (Matsunami Glass Ind. Ltd., Osaka, Japan) and covered with coverslips. A histopathologist observed changes using a Microphot-FX (Nikon) in a blinded manner. Microphotographs were taken using a Digital Sight DS-5M camera (Nikon) equipped with a microscope.
6
Fungal Growth on Various Agar Media
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The fungus grew on culture plates of malt extract ager (MEA; Oxoid, U.K.), oatmeal agar (OA; home-made at CBS), potato dextrose agar (PDA; Oxoid), synthetic nutrient agar (SNA) [15 ], and carnation leaf agar (CLA) [16 ]. Culture plates were incubated in the dark for one week at 25°C. Microscopic mounts in lactic acid with cotton blue were made from cultures grown on a PDA plate. Slide cultures were observed after 5 days of incubation at 25°C. Slides were examined and measured with a light microscope (Nikon Eclipse 80i), and pictures were taken using a Nikon digital-sight DS-5 M camera attached to the microscope.
7
Morphological Characterization of Fungal Cultures
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Cultures preserved in the CBS-KNAW, Utrecht, The Netherlands were used for the present study. Cultures were activated from lyophilised or cryopreserved material and inoculated on oatmeal (OA) and 3 % malt extract (MEA, Oxoid) agars, prepared according to Crous et al. (2009) . For culture studies, 5-d-old cultures were transferred to fresh plates and incubated in the laboratory in diffuse daylight (20 °C), and in an incubator under n-UV light (12 h light, 12 h dark) at 18 °C to promote sporulation. Colony diameter measurements were taken from OA plates placed in the incubator with UV, after 10 d. Colours were described according to Rayner (1970) . Sporulating structures obtained from cultures were used for the morphological description. Structures were mounted in water and examined with an Olympus BX 50 microscope mounted with bright field and differential interference contrast (DIC) objectives, and photographed using a mounted Nikon Digital Sight DS-5M camera. Photographs of culture plates were taken after 10 and 14 d on a photo stand with daylight tubes with a Pentax K110 D digital camera. Conidial masses from OA plates were mounted in water and 30 spores measured. Length/width (L/W) ratio was calculated for each spore and average L/W ratio calculated (N = 30). Descriptions and nomenclature of taxonomic novelties were deposited in MycoBank (www.MycoBank.org ; Crous et al. 2004 ).
8
Immunofluorescent Staining for PCNA
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For immunofluorescent staining for PCNA (proliferation) samples were incubated overnight at 4 °C with anti-human PCNA antibodies (Dako, dilution 1:200) and secondary Alexa 488-conjugated antibodies (Invitrogen, dilution 1:200). Slides were mounted in a Vectashield medium (Vector Labs) containing DAPI dye. Samples were photographed using a Nikon Eclipse TS100 microscope equipped with a Nikon Digital Sight DS-5 M camera. Images were analyzed with GSA Image Analyzer software.
9
Immunohistochemical Detection of ODCrp[A] in Kidney
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Five-micrometer thick sections from formalin fixed and paraffin-embedded kidneys were stained with 1:1200 diluted rabbit anti-ODCrp[A] antibody and with its preimmune serum as control using Vectastain Elite ABC Kit (Vector Laboratories Inc., Burlingame, U.S.A.) according to the manufacturer’s instructions essentially as described [29 (link)]. Light microscope photographs were taken with an Olympus BX51 microscope (Olympus Optical, Tokyo, Japan) and a Nikon Digital Sight DS-5M camera (Nikon Corporation, Tokyo, Japan) using NIS-Elements F2.30 software (Nikon Corporation). Digital image processing was performed with PhotoScape v3.6.1 (Mooii Tech, Informer Technologies Inc., Los Angeles, U.S.A.).
10
Immunofluorescence Staining of GFAP and IBA-1
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Each section was treated with 4% paraformaldehyde in PBS for 1 h at room temperature, then washed twice with PBS and incubated in 2 M HCl at 37°C for 1 h for immunofluorescence staining. The sections were subsequently incubated in blocking solution with mouse monoclonal anti-GFAP (1 : 1,000; EMD Millipore, Billerica, MA, USA) or rabbit polyclonal anti-IBA-1 primary antibodies (1 : 50; Abcam, Cambridge, UK) overnight at 4°C. The sections were then incubated with Alexa Fluor-488 goat anti-mouse immunoglobulin G (IgG; 1 : 200; Invitrogen; Thermo Fisher Scientific Inc.) and DyLight 549 anti-rabbit IgG (1 : 200; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) secondary antibodies at room temperature for 2 h. The sections were counterstained with DAPI and mounted with Mounting Medium H-1000 (Vector Laboratories Inc., Burlingame, CA, USA). Negative control was established by omitting the primary antibodies. Fluorescent microscopy images were obtained using a Nikon ellipse 80i microscope (Nikon Corporation, Tokyo, Japan) and a Nikon Digital Sight DS-5M camera, using the NIS-Elements F 2.30 software (Nikon Corporation). Digital image processing was performed by Image-Pro Plus, version 5.1.
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