Specimens of Paurodontella composticola n. sp. were obtained from compost collected in Nazar Abad City, Alborz Province, Iran in August 2017. To obtain a cleaner suspension of nematodes, the tray of extraction was employed (Whitehead and Hemming, 1965). Specimens for light microscopy were killed by gentle heat, fixed in a solution of 4% formaldehyde + 2% glycerol and transferred to anhydrous glycerin, according to De Grisse (1969) , and mounted on permanent slides. Specimens were examined using an Olympus BH-2 (Japan) compound microscope at magnifications up to 1,000× magnification. Measurements were carried out using a drawing tube attached to a Nikon E200 light microscope (Japan). All measurements were expressed in micrometers (μm) and photographs of nematodes were taken by a digital camera attached with the same microscope.
E200 light microscope
Manufactured by Nikon
Sourced in Japan
The Nikon E200 light microscope is a laboratory equipment designed for optical observation and analysis. It features a compound optical system that utilizes transmitted illumination to magnify and examine samples. The E200 can provide high-quality, detailed images of a variety of specimens.
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8 protocols using e200 light microscope
1
Paurodontella composticola n. sp. Isolation and Examination
2
Histological Analysis of Organ Tissues
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After the animals died, the heart, liver and kidneys were removed carefully and immersed in 10% formaldehyde at room temperature and then sectioned transversely into 5μm slices. Specimens were dehydrated in a graded series of alcohol and xylene and embedded in paraffin. Multiple slices (15 fields for each slide) were made and stained by hematoxilin and eosin stains. Sections were viewed and were photographed using a Nikon E200® light microscope (Japan).
This study was approved by Ethics Committee of this institute. Razi Vaccine and Serum Research Institute.
This study was approved by Ethics Committee of this institute. Razi Vaccine and Serum Research Institute.
3
Testicular Cell Quantification using Optical Disector
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Optical disector method and unbiased computer-generated frames were employed for counting all nuclei and final estimation of the total number of individual cells in each testis [23 (link)]. The procedure was done with a Nikon E200 light microscope (Tokyo, Japan) fitted with a 60x oil objective lens at a final magnification of × 1640 and a microcator (MT12, Heidenhain, Traunreut, Germany) used to monitor field depth. The numerical density (Nv) of the cells was calculated as follows:
Nv (cells) = [ΣQ / (ΣP × (a/f) × h)] × (t/BA).
Where ΣQ is the number of each cell type nuclei, a/f is the area per counting frame, ΣP is the total number of counted frames per animal; h is the height of the optical disector measured using the microcator, t is the mean thickness of final sections, and BA is the microtome block advance. The total number of favored cells per testis [N (cells)] were estimated by multiplying the numerical density (Nv) by the reference volume:
N (cells) = NV (cells) × V (structure).
Where V (structure) is the total volume of germinal epithelium for the cells in the germinal layer, and the total volume of interstitial tissue for Leydig cells.
Nv (cells) = [ΣQ / (ΣP × (a/f) × h)] × (t/BA).
Where ΣQ is the number of each cell type nuclei, a/f is the area per counting frame, ΣP is the total number of counted frames per animal; h is the height of the optical disector measured using the microcator, t is the mean thickness of final sections, and BA is the microtome block advance. The total number of favored cells per testis [N (cells)] were estimated by multiplying the numerical density (Nv) by the reference volume:
N (cells) = NV (cells) × V (structure).
Where V (structure) is the total volume of germinal epithelium for the cells in the germinal layer, and the total volume of interstitial tissue for Leydig cells.
4
Histological Analysis of Bone-Implant Interface
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All samples were immediately immersed in 10% buffered formalin and kept in this solution for 7 days. Then, they were dehydrated in an ethanol solution sequence (50–100%) and embedded in a historesin (Technovit 7200 VLC, Kulzer, Wehrheim, Germany). The cuts were performed using an IsoMet 1000 machine (Buehler, Lake Bluff, USA). The slides were stained using the picrosirius–hematoxylin technique33 . Images were obtained using a Nikon E200 light microscope (Tokyo, Japan). For histological measurements, ImageJ for Windows™ software was used (National Institute of Health, Bethesda, USA). The percentage of contact between bone and implant (BIC%) was measured along the entire length of the implant surface.
5
Histomorphometric Analysis of Bone Healing
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Images of the slides were taken at different sizes using a Nikon E200 light microscope (Tokyo, Japan). Initially, all histological sections of each sample were qualitatively evaluated, describing the characteristics of the healing reaction at each proposed time. Then, a histomorphometric analysis was performed to evaluate the following aspects of the materials and healing events: (1) fibrous tissue (FB);
(2) newly formed bone matrix (BM); (3) medullary spaces (MS); and (4) remaining material particles (RP). All these parameters were measured in three predetermined areas of 1 mm 2 in each defect, as shown in Figure 2. The values were calculated as the percentage occupancy in relation to the total area.
To facilitate statistical comparison, and because they present similar conditions for healing, from the values measured at Positions 1 and 3, an average was calculated for each group at each proposed evaluation time.
In addition, the visible area in each histological section was measured, as shown in Figure 3. All measurements were performed using ImageJ version 1.44 software (National Institute of Mental Health, Bethesda, Maryland, USA).
(2) newly formed bone matrix (BM); (3) medullary spaces (MS); and (4) remaining material particles (RP). All these parameters were measured in three predetermined areas of 1 mm 2 in each defect, as shown in Figure 2. The values were calculated as the percentage occupancy in relation to the total area.
To facilitate statistical comparison, and because they present similar conditions for healing, from the values measured at Positions 1 and 3, an average was calculated for each group at each proposed evaluation time.
In addition, the visible area in each histological section was measured, as shown in Figure 3. All measurements were performed using ImageJ version 1.44 software (National Institute of Mental Health, Bethesda, Maryland, USA).
6
Histopathological Analysis of Liver and Lung Tissues
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Liver and lung tissues were either frozen quickly or fixed with 4% paraformaldehyde for more than 24 h. Hematoxylin and eosin (H&E) staining was performed in fixed samples and sections which were cut at a thickness of 5 μm. Lipid droplet accumulation in the liver was visualized using Oil red O (O0625; Merck, Darmstadt, Hesse, Germany) staining of frozen liver sections that were prepared in optimum cutting temperature (O.C.T.) compound (4583; Tissue-Tek, Sakura). Histopathology images were captured with a Nikon E200 light microscope (Nikon, Tokyo, Japan).
7
Matrigel Cell Invasion Assay Protocol
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Matrigel cell invasion assays were performed using 8‐μm polycarbonate membranes (Corning, Acton, MA, USA) according to the manufacturer's instructions. Cell suspension (5 × 105 cells; 100 μl) was added to the upper chamber, while the lower chamber was filled with RPMI‐1640 medium containing 10% foetal calf serum. The plates were incubated for 24 hrs, and then cells that had invaded to the lower membrane surface were fixed in methanol for 30 min. and stained with haematoxylin (Sigma). For each filter, the numbers of cells that invaded to the lower surface of the membrane were counted in five randomly selected fields of view at 200 × magnification using a Nikon E200 light microscope (Nikon, Melville, NY, USA). Mean cell numbers were calculated from the data obtained from each experiment repeated three times.
8
Immunohistochemical Analysis of Periapical Lesions
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Immunohistochemical analysis was performed by two oral pathologists with a Nikon E200 light microscope (Nikon, Tokyo, Japan). Tissue sections were examined under light microscopy (100X magnification) to identify areas containing the largest number of immunoreactive cells, and the selected microscopic fields were then analyzed at 400X magnification. The following aspects were considered in evaluating the TGF-β1 and MMP-9 immunostaining: presence (+) or absence (-) of immunostaining, type of immunopositive cells and their tissue distribution (focal or diffuse). Immunoexpression scores were attributed semi-quantitatively to the percentage of positive cells in each case: score 0 -no positive cells; score 1 -1-50% positive cells; and score 2 -> 50% positive cells.
A descriptive statistical analysis was conducted using Windows Office Excel 2015®. WinPepi software for Windows, version 11.32, 2013, was used to test the possible differences and/or inferential statistical associations between the immunoexpression scores in periapical granulomas and radicular cysts, using the non-parametric Mann-Whitney, Pearson´s chi-square, Fisher's exact tests and Spearman's correlation test. The confidence interval was defined as 95% and a value of p < 0.05 was considered statistically significant.
A descriptive statistical analysis was conducted using Windows Office Excel 2015®. WinPepi software for Windows, version 11.32, 2013, was used to test the possible differences and/or inferential statistical associations between the immunoexpression scores in periapical granulomas and radicular cysts, using the non-parametric Mann-Whitney, Pearson´s chi-square, Fisher's exact tests and Spearman's correlation test. The confidence interval was defined as 95% and a value of p < 0.05 was considered statistically significant.
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