Digoxigenin-11-UTP-labeled single-stranded RNA probes for murine Lef1, Bmp4, Msx1, Fgf9, Fgf10, Fgf4, Epfn, Dkk1, Wnt10b, and Shh were prepared using a DIG RNA labeling kit (Roche Applied Science) according to the manufacturer's instructions. For whole-mount in situ hybridization, E11.5 branchial arches and E14.5 and E17.5 first molars cultured with 20 μM BIO or MetBIO for 24 or 48 h were fixed in 4% paraformaldehyde/PBS overnight, dehydrated in methanol, and kept at −20°C until use. Whole-mount RNA in situ hybridization was carried out according to Nieto et al. (1996 (link)). Some hybridized tissues were sectioned at 15 μm using a Shandom cryostome. Images were obtained with a Zeiss Stemi 2000-C stereomicroscope equipped with a Canon PowerShot A80. The number of the samples (n) for the in situ hybridization was 10 for each experimental condition, both control and BIO. Hybridization for each probe was performed at least twice.
Stemi 2000 c stereomicroscope
Manufactured by Canon
The Stemi 2000-C is a stereomicroscope designed for observing and analyzing samples. It provides a three-dimensional view of the specimen, allowing for detailed examination. The microscope features optical components that enable high-resolution imaging and magnification capabilities.
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3 protocols using stemi 2000 c stereomicroscope
1
Murine Craniofacial Development Molecular Profiling
2
Insect Collecting and Morphological Analysis
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The specimens were collected in northwestern China from July to August 2016–2017 using sweep netting, light trapping, and Malaise trapping. The specimens were studied and illustrated with a ZEISS Stemi 2000-c stereomicroscope; the photo illustrations were taken under a Canon Mark IV with Canon MP-E 65 mm lens. The descriptions were based on specimens preserved in 95% alcohol. Genitalic preparations of males were made by macerating the apical abdomen in cold 10% NaOH for 12–15 hours. After examination, preparations were transferred to fresh glycerin for preservation and stored in a microvial pinned below the specimen. The morphological terminology mainly follows McAlpine (1981) . Type specimens are deposited in the Entomological Museum of China Agricultural University (CAU
3
Developmental Staging and Pupation Timing Analysis
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Developmental staging was performed as previously described [33] . Early third-instar larvae (0-3 h after the second ecdysis) were defined as E3rd. Pupal stage was defined as hours after puparium formation (APF) by collecting newly formed white pupae (WP or 0 h APF).
For the analysis of pupation timing, newly formed white pupae were collected and placed in a 10 cm culture dish. Rolled pieces of wetted Kimwipe were placed on the side of the dish to create a humid chamber. Time-lapse images were acquired at 10-min intervals using a Canon EOS Kiss X7 digital camera in an incubation chamber. Pupation was judged as the time at which head eversion begins after detachment from pupal case. Experiments were repeated with at least four independently rared populations, and all data were summed.
Lethal phase during the pupal stage was determined under a stereomicroscope (Zeiss) in at least two independent experiments with more than four vials in total. The presented graphs are the sum of all data. Pupae were photographed under a Zeiss Stemi 2000-C stereomicroscope equipped with a Canon PowerShot G15 digital camera.
For the analysis of pupation timing, newly formed white pupae were collected and placed in a 10 cm culture dish. Rolled pieces of wetted Kimwipe were placed on the side of the dish to create a humid chamber. Time-lapse images were acquired at 10-min intervals using a Canon EOS Kiss X7 digital camera in an incubation chamber. Pupation was judged as the time at which head eversion begins after detachment from pupal case. Experiments were repeated with at least four independently rared populations, and all data were summed.
Lethal phase during the pupal stage was determined under a stereomicroscope (Zeiss) in at least two independent experiments with more than four vials in total. The presented graphs are the sum of all data. Pupae were photographed under a Zeiss Stemi 2000-C stereomicroscope equipped with a Canon PowerShot G15 digital camera.
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