Specimens were examined on a Leica Confocal imaging system, an Olympus FV1000 Confocal microscope, a Leica epifluorescence microscope or a Leica EZ4D microscope. For image analysis we used the Image J software [25 ]. All images from each sample were acquired under the same conditions (exposure, brightness, gamma levels). For graft reconstructions the photographs were taken with a Leica Confocal imaging system, and the reconstruction was performed manually only for the site of the graft.
Ez4d microscope
Manufactured by Leica
Sourced in Switzerland, Japan
The EZ4D microscope is a compact and versatile digital microscope designed for a wide range of applications. It features a high-resolution CMOS camera and a built-in 5-inch LCD display for convenient viewing and image capture. The EZ4D microscope offers a continuous magnification range, allowing users to seamlessly adjust the magnification as needed for their specific tasks.
Automatically generated - may contain errors
7 protocols using ez4d microscope
1
Confocal Imaging Protocol for Tissue Reconstruction
2
Quantifying Dopaminergic Fiber Density
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The density of TH+ fibers in the striatum was calculated as previously described [30 (link)], using three to four coronal brain sections from each rat. Striatal sections were processed for TH immunohistochemistry as described above. Images were taken with a Leica EZ4D microscope with constant illumination and equal optical parameters were used for all acquired images. Digitized images were analyzed with ImageJ software. The data are presented as optical density (O.D.) values for each hemisphere. In cases where the O.D. of a lesioned striatum was higher than expected, we corroborated cell loss at the SNpc directly by counting TH+ cell bodies in that brain structure.
3
Quantifying Achilles Tendon Adhesion Formation
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After extraction, the Achilles tendon specimens were immediately frozen at −20°C. After being thawed to room temperature, they were dehydrated, paraffin-embedded and sectioned into 5 µm thick slices, which were cross-sections in the wound region (perpendicular to the Achilles tendon). After de-paraffinizing with xylene and rehydrating the sections, they were stained with Hematoxylin-Eosin (H&E), Alcian Blue (AB) and Picrosirius Red (PS) according to commonly established procedures.
Picrosirius Red stained sections were used to quantify the adhesion extent at 8× magnification (Leica EZ4D microscope, Switzerland). Here, adhesion formation was quantified in five subsequent cross-sections separated by 2.0 mm using a method by Tan et al. (2010) (link). The percentage of adhesion was calculated by the length of the contact region of the tendon under view with the surrounding tissue divided by the total perimeter. The length of the contact region and the whole perimeter were determined using synedra View 3 software (version 3. 1. 0. 3.).
Picrosirius Red stained sections were used to quantify the adhesion extent at 8× magnification (Leica EZ4D microscope, Switzerland). Here, adhesion formation was quantified in five subsequent cross-sections separated by 2.0 mm using a method by Tan et al. (2010) (link). The percentage of adhesion was calculated by the length of the contact region of the tendon under view with the surrounding tissue divided by the total perimeter. The length of the contact region and the whole perimeter were determined using synedra View 3 software (version 3. 1. 0. 3.).
4
Measuring Weights and Anogenital Distance
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Body weights of C- and R-females/fetuses/offspring were measured with a portable balance (EK-3000i) (A&D, Tokyo, Japan), and testis weights of C- and R-offspring were measured with an analytical electronic balance (AX124) (Sartorius AG, Göttingen, Germany). AGD was measured with a Digimatic Caliper (Mitsutoyo, Kawasaki, Japan) under a EZ4 D microscope (Leica, Wetzlar, Germany). The AGD index (AGDI) was also calculated by diving the AGD by the cube root of body weight [17 (link)], to allow for comparison of AGDs between C-fetuses and R-fetuses with different body size.
5
Fruit Fly Species Identification
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Taxonomic keys by White and Elson-Harris (1992 ), De Meyer (1996 (link), 1998 (link), 2000 (link)), Billah et al. (2007 ), and were used with aid of a Lecia EZ4 D microscope to identify the various species of fruit flies collected from the traps. The identification was done in the African Postgraduate Programme in Insect Science (ARPPIS) Laboratory. Confirmation and inability to identify species were referred to M.K. Billah, a Fruit Fly Taxonomist in the Department of Animal Biology and Conservation Science, University of Ghana, Legon.
6
Visualizing AtMyb56 Promoter Activity
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A GUS staining solution consisting of 0.5 mM potassium ferricyanide, 10 mM EDTA, 100 mM sodium phosphate buffer (pH 7.0), 0.5 mM potassium ferrocyanide, 1 mM X-Gluc, and 0.1% Triton X-100 was manufactured. Seedlings were soaked in the GUS solution at 37°C for 1 h and chlorophyll removed by subsequent incubation in 70% ethanol for 6 h. Seedlings were then observed and photographed under a Leica EZ4D microscope (Jefferson et al., 1987 (link)). To generate AtMyb56pro:GUS reporter lines, a 1-kb promoter region of AtMyb56 was amplified with the primers of AtMyb56 F (5′-CGT TAT GGA GAG AAC AGA AAG CTC-3′) and R (5′-GTT TCT GGG TTT AGG GAT TAA GG-3′), inserted into the TOPO vector using a pCR8/GW/TOPOR TA CloningR Kit vector (Invitrogen™), and then transferred into the pMDC162 vector (Curtis and Grossniklaus, 2003 (link)). DNA sequencing was analyzed to confirm the integrity of this construct prior to the transformation of Arabidopsis (Columbia ecotype) via the Agrobacterium tumefaciens-mediated floral dipping method (Clough and Bent, 1998 (link)).
7
Seedling Morphology and Anatomy Analysis
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To obtain the seedlings, the seeds were pulped by manual maceration with the aid of a sieve, disinfected with sodium hypochlorite, washed twice in running water and placed in Petri dishes with 3 moistened filter paper and left at a temperature of 25ºC in a germination chamber. with a period of 12h until the protrusion of the primary root. The germinated seeds were transferred to a polystyrene tray with vermiculite, kept in a greenhouse and irrigated four times a week. The morphological analysis of the seedlings was based on the terminology of Rizzini (1977) , Vogel (1980 ), Garwood (1996) and Souza et al., (2009) .
Anatomical analysis of seedlings was performed on samples fixed in glutaraldehyde (1%) + formaldehyde (4%) and FAA (formaldehyde, acetic acid and ethanol) 50, embedded in historesin (Leica®), sectioned in a rotation microtome and stained with toluidine blue (O'BRIEN et al., 1964) .
Samples were also sectioned freehand and stained with safranin and astra blue. The anatomical illustrations were made with photomicrographs obtained by image capture in a digital camera coupled to a Leica EZ4D microscope, whose images were processed using Leica Application Suite Version 1.8 software.
Anatomical analysis of seedlings was performed on samples fixed in glutaraldehyde (1%) + formaldehyde (4%) and FAA (formaldehyde, acetic acid and ethanol) 50, embedded in historesin (Leica®), sectioned in a rotation microtome and stained with toluidine blue (O'BRIEN et al., 1964) .
Samples were also sectioned freehand and stained with safranin and astra blue. The anatomical illustrations were made with photomicrographs obtained by image capture in a digital camera coupled to a Leica EZ4D microscope, whose images were processed using Leica Application Suite Version 1.8 software.
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