Antisense riboprobes were synthesized and in situ hybridization was performed as previously described [29 (link)], with the addition of 5% dextran sulfate (Sigma-Aldrich D8906) to the hybridization buffer. To increase visualization of staining, some 24 hpf embryos were also incubated in 3%H2O2/0.5%KOH to remove pigmentation. Post-hybridization washes were carried out using a Biolane HTI in situ machine (Huller and Huttner AG). After staining, embryos were stored in MeOH and cleared in 70% glycerol for dissection and imaging. For sectioning, embryos were post-fixed in 4% paraformaldehyde for 1–2 hours at room temp, washed in PBS, and cryoprotected in 30% sucrose in PBS. Embryos were then embedded in OCT and sectioned at 20μm thickness on a Leica cryostat. Images were acquired using an Olympus BX51WI compound microscope and an Olympus Microfire camera. Digital images were cropped and aligned using Adobe Photoshop.
Microfire camera
Manufactured by Olympus
Sourced in United States
The Microfire camera is a compact and versatile imaging device designed for laboratory and research applications. It provides high-quality image capture capabilities for a range of microscopy and analysis tasks. The camera features a state-of-the-art image sensor and advanced image processing capabilities to deliver clear and detailed visual data.
Automatically generated - may contain errors
Lab products found in correlation
Bx51wi compound microscope, by Olympus (2 mentions)
Cm3050s cryostat, by Leica (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
Cryostat, by Leica (1 mentions)
D8906, by Merck Group (1 mentions)
Ix70 inverted microscope, by Olympus (1 mentions)
Inverted phase microscope, by Olympus (1 mentions)
Cm3050 cryostat, by Leica (1 mentions)
7 protocols using microfire camera
1
In Situ Hybridization and Embryo Imaging
2
Histological Verification of RVM and PB Injections
3
Automated Nuclear Counting Using ImageJ
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All images were captured using a MICROfire camera (Olympus, Center Valley, PA) attached to an Olympus IX70 inverted microscope using either a 10× or 20× objective. Images were merged and colored using ImageJ (US National Institutes of Health, Bethesda, MA). To remove human bias, ImageJ was used for the automated counting of Hoechst-labeled nuclei. Images underwent thresholding to remove background and were then converted to binary black and white images. The “Analyze Particles” function was used to count the nuclei, excluding any small (less than 600 pixels) punctate nuclei to prevent apoptotic Hoechst+ nuclear debris from being counted.
4
In Vitro Wound Healing Assay
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Cells were plated at confluence in 24-well plates. A wound was created by scraping the confluent monolayer with a p200 pipette tip, and cells were grown in medium supplemented with 1% FBS. The migration of the cells was recorded at 0, 24, and 48 h using a MicroFire camera fitted to an Olympus Inverted Phase Microscope (Olympus) at 10X magnification. The area of the uncovered wound gap was measured using ImageJ software, and details are described in our previous studies (27 (link), 28 (link)). All experiments were repeated in triplicate.
5
Histological Identification of RVM
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At the end of each experiment, the recording site was marked with an electrolytic lesion. Animals were euthanized by methohexital overdose and perfused transcardially with saline and 10 % formalin. Brains were removed, and brainstems were sectioned on a Leica CM3050 S cryostat (60 µm sections). RVM lesion was photographed with an Optronics Microfire camera attached to an Olympus BX51 microscope. The RVM was defined as the nucleus raphe magnus and adjacent reticular formation medial to the lateral boundary of the pyramids at the level of the facial nucleus (Paxinos and Watson, 2009 ).
6
Zebrafish Embryo Microinjection Protocol
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Wild-type AB strains of Danio rerio (D. rerio- zebrafish) embryos were supplied from the zebrafish core facility center, National Tsing Hua University. The MNDC samples were ultrasonicated for 30 min in Milli-Q water and then microinjected (10 nL) in the fresh embryos placed on the microinjection embryo tray. The dark incubated (28 °C) control embryos (without MNDC) and MNDC-loaded embryos were periodically observed using microscopic images49 (link),50 (link). The abnormality of infant fish was detected using a MicroFire camera (Olympus CX-21i Trinocular Microscope, LED illumination) mounted onto a Leica MZ16 stereomicroscope (Meyer Instruments, Houston, TX, USA).
7
In situ Hybridization of Embryo Sections
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