Mating assays were performed on Murashige-Skoog media (Sigma, USA) and V8 juice media plus 100 μg/mL myo-inositol (pH 5.0) (Sigma, USA) using KN99a as the MATa partner. Strains were co-inoculated in equal quantities via toothpick and incubated in the dark at room temperature for two weeks. Filamentation and sporulation was examined and imaged using a Leica M125 stereomicroscope fitted with a Leica DFC 425c digital camera running Leica Application Suite 3.6 software (Leica, Germany).
Application suite 3
Manufactured by Leica
Sourced in Germany
Application Suite 3.3 is a software package developed by Leica for their lab equipment. It provides core functionalities for image acquisition, processing, and analysis. The suite offers a range of tools to support various laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
M205 fa, by Leica (2 mentions)
Dfc7000 t camera, by Leica (2 mentions)
Dfc345 fx camera, by Leica (2 mentions)
Dm6000b microscope, by Leica (2 mentions)
Murashige skoog media, by Merck Group (1 mentions)
Dfc425c, by Leica (1 mentions)
Myo inositol, by Merck Group (1 mentions)
S8apo, by Leica (1 mentions)
Rapamycin, by Cayman Chemical (1 mentions)
Optiphot 2 microscope, by Nikon (1 mentions)
Dm2500, by Leica (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
13 protocols using application suite 3
1
Mating Assay for Fungal Strains
2
Evaluating Irrigant Penetration Depth
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Initially, a total volume of 2 mL of contrast solution was delivered and left in the canals using the 30-G NaviTip needle. The EasyClean file was mounted on a VDW Silver motor, and the tip was passively inserted 3 mm from the WL and activated in rotary motion at 1000 rpm. This procedure was repeated three times. The total irrigant volume for each canal was 6 mL, and the total activation time was 1 min.
After the activation procedures, a round silicone base with a rectangular slot fitting the microscope base was positioned under a steromicroscope (S8 APO, Leica, Wetzlar, Germany) connected to a digital camera (CMOS 10 megapixels, Opticam, São Paulo, SP, Brazil). The rectangular slot matched the exact dimensions of the resin blocks. The digital image of each specimen was captured using Leica Application Suite 3.6 (Leica) at 1.25× magnification and was saved in TIFF.
Then, one trained evaluator, blinded to the group assignment of each sample, analysed the images using Adobe Photoshop CS6 Extended software (Adobe Systems Inc., San Jose, CA, USA). The extension of the irrigant penetration was measured in mm for each lateral canal. Data were analysed using BioEstat (MCT-CNPq, Belém, PA, Brazil), version 5.0. The difference between the groups was compared using one-way analysis of variance followed by Tukey’s post hoc test (P<0.05).
After the activation procedures, a round silicone base with a rectangular slot fitting the microscope base was positioned under a steromicroscope (S8 APO, Leica, Wetzlar, Germany) connected to a digital camera (CMOS 10 megapixels, Opticam, São Paulo, SP, Brazil). The rectangular slot matched the exact dimensions of the resin blocks. The digital image of each specimen was captured using Leica Application Suite 3.6 (Leica) at 1.25× magnification and was saved in TIFF.
Then, one trained evaluator, blinded to the group assignment of each sample, analysed the images using Adobe Photoshop CS6 Extended software (Adobe Systems Inc., San Jose, CA, USA). The extension of the irrigant penetration was measured in mm for each lateral canal. Data were analysed using BioEstat (MCT-CNPq, Belém, PA, Brazil), version 5.0. The difference between the groups was compared using one-way analysis of variance followed by Tukey’s post hoc test (P<0.05).
3
Embryo Deformity Assessment at 120 hpf
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Embryo mortality was determined on the basis of egg coagulation, the lack of somite formation, and the lack of heart function at 120 hpf. Sublethal endpoints (pericardial edema, yolk edema, tail deformation, craniofacial deformation, and disintegrated abnormal embryo shape) were also determined at 120 hpf. Abnormalities were examined separately, irrespective of the number of deformities in the individual. Digital images were captured of laterally oriented 120 hpf larvae at 30× magnification using a stereomicroscope (Leica M205 FA, Leica DFC 7000T camera, Leica Application Suite 3.4.2.18368, Leica Microsystems GmbH, Wetzlar, Germany).
4
Zebrafish Embryo Microinjection Protocol
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One-cell stage zebrafish embryos were lined up against the side of a microscope slide placed in a 10 cm diameter Petri dish. Excess water was removed with a plastic pipette. Treatment groups of 20 eggs were injected in a minimum of three replicates per treatment. Following microinjection, eggs were incubated in system water with methylene blue (2 mL 0.1% methylene blue in 1 L system water) (25°C ± 2°C) in 10 cm diameter Petri dishes. After 2 hours, coagulated and/or non-fertilized eggs were discarded and developing embryos were transferred in groups of twenty into 6 cm diameter Petri dishes. Embryos were then incubated in system water at 26°C ± 1°C and a 14 h:10 h-light:dark period and checked for lethal and sublethal effects under a microscope. System water was replaced in every 24 hours until 120 hpf (hours post-fertilization). Digital images of embryos (72 hpf) and larvae (120 hpf) in lateral orientation were taken under a stereomicroscope at 30× magnification (Leica M205 FA, Leica DFC 7000T camera, Leica Application Suite 3.4.2.18368, Leica Microsystems GmbH, Germany).
5
Transgenic Plant Staining Visualization
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Transgenic plants obtained as mentioned above were stained as described (Jefferson et al., 1987 ). Images were captured with a stereoscope (Leica Application Suite 3.3, Germany)
6
Sporulation and Rapamycin Treatment Protocol
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All analysis was carried out using the SK1 (can1), diploid strain (ATCC). Cultures were routinely grown in YPD (1% Bacto-yeast extract, 2% Bacto-peptone, 2% glucose) to the required cell density.
For sporulation experiments, one single colony was inoculated in YPD and grown to 2x107cell/ml. After centrifugation and washing with sterile water, the pellet was resuspended to a 5x106cell/ml density in PSP2 medium [30 (link)]. The culture was grown for five generations at 28–30°C with vigorous shaking, and then harvested and washed with water, followed by resuspending in SPM (0.3% potassium acetate, 0.02% raffinose) [30 (link)] to a 107cell/ml density to induce sporulation. Following 3 hours vigorous shaking at 28–30°C, the culture was spun down and the pellet was used for RNA extraction, or polysome profiling.
For the rapamycin treatment, SK1 or ime4Δ/ ime4Δ cells were grown to a late log phase in YPD and rapamycin was added to a final concentration of 200ng/ml (Cayman Chemical). Following the treatment the cultures were monitored and processed at defined time intervals.
Microscopic images were generated by a Leica DFC320 camera and NIKON OPTIPHOT-2 microscope (400x magnification). Images were processed in the Leica Application Suite 3.3.
For sporulation experiments, one single colony was inoculated in YPD and grown to 2x107cell/ml. After centrifugation and washing with sterile water, the pellet was resuspended to a 5x106cell/ml density in PSP2 medium [30 (link)]. The culture was grown for five generations at 28–30°C with vigorous shaking, and then harvested and washed with water, followed by resuspending in SPM (0.3% potassium acetate, 0.02% raffinose) [30 (link)] to a 107cell/ml density to induce sporulation. Following 3 hours vigorous shaking at 28–30°C, the culture was spun down and the pellet was used for RNA extraction, or polysome profiling.
For the rapamycin treatment, SK1 or ime4Δ/ ime4Δ cells were grown to a late log phase in YPD and rapamycin was added to a final concentration of 200ng/ml (Cayman Chemical). Following the treatment the cultures were monitored and processed at defined time intervals.
Microscopic images were generated by a Leica DFC320 camera and NIKON OPTIPHOT-2 microscope (400x magnification). Images were processed in the Leica Application Suite 3.3.
7
Promoter analysis of FLO19 in rice
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The putative promoter region of FLO19 (2271 bp upstream of ATG) was amplified by PCR and inserted into the BamHI/HindIII sites of pCAMBIA1381Z. The binary vector was introduced into Nipponbare callus using Agrobacterium tumefaciens‐mediated transformation. After PCR screening of the resulting transgenic plants, 10 positive lines were identified and T2 transgenic progeny were used for GUS staining (Jefferson et al., 1987 (link)). Images of various tissues were obtained with a stereoscope (Leica Application Suite 3.3, Germany).
8
Histopathological Analysis of Infected Tissues
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The gastrocnemius muscle and footpad were collected at defined days post infection and fixed with 4% of formaldehyde for 24 h. The footpad was decalcified using EDTA solution (125 g/L, pH 7.0) and then fixed. Tissues were embedded in paraffin after dehydration. Paraffin-embedded tissue sections of 5 μm were prepared and stained with hematoxylin and eosin (H&E). Images were obtained using optical microscopy with a magnification of 10 X (Olympus BX40), and images were acquired using software Leica Application Suite 3.8 (Leica).
9
Acridine Orange Staining of TiO2 NPs Exposed Onion Root Tips
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The onion root tips were interacted with the TiO2 NPs solution for 4 h. About 2 Root tips were then taken out and incubated for 2 min in the presence of 1 N HCl at room temperature. Tips were stained with nuclear specific stain acridine orange (AO) for visualization of chromosomal aberrations. To the root tips treated with 1 N HCl, 20 µL of AO (15 µg/mL in PBS) was added and incubated for 15 min under dark conditions. The suspension was then centrifuged at 6000 rpm for 10 min at 0°C, and the supernatant was discarded to eliminate unbound dyes after 5 min of incubation. The root tips were resuspended with Millipore filtered water. This was repeated three times in order to ensure complete removal unbound dyes. The root tips were placed on to a glass slide and covered with cover slip. Care was taken to avoid formation of air bubbles while placing the cover slip. Finally, the cover slip was pressed firmly with the help of thumb to prepare a uniform squash of the root tips. Dark condition was maintained throughout to avoid photo bleaching of dyes. Fluorescence images were observed using the BP 450–490, LP 590 filter using a fluorescence microscope (DM-2500, Leica, Germany). The images were recorded with the attached camera component (Leica-DFC-29) and processed using Leica-Application Suite 3.8.
10
Chromosomal Analysis of Mapuchea chilensis
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Four adult males of Mapuchea chilensis 40.6667°S, 72.1742°W ) on 15–17 January 2014 from leaf litter between creeping stems of Hydrangea serratifolia
The classification of cicadomorphanAuchenorrhyncha
The classification of cicadomorphan
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