Twelve embryos from the uninjected group, 11 from the control-MO injected group, and 14 from the Snrnp200-MO injected group were harvested and cryopreserved per standard procedures. They were fixed in 4% paraformaldehyde, incubated with 30% sucrose, embedded with optimal cutting temperature solution, and frozen in liquid nitrogen for sectioning at 5 μm using a Leica CM1900 cryostat (Leica, Wetzlar, Germany). Rod and cone photoreceptors were further visualized through immunofluorescence staining as indicated previously [12 (link)]. Briefly, cryosections were incubated with designated primary antibodies, including antirhodopsin (Mouse, 1 : 250; Abcam, Cambridge, UK) and zpr-1 antibodies (Mouse, 1 : 250; ZRIC, USA), to label rod and cone photoreceptors, respectively. The cryosections were then treated with fluorescence-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) for another 1 hr at room temperature and finally counterstained by 4′,6-diamidino-2-phenylindole (DAPI; Sigma, USA) for cell nuclei staining. Images were taken with an Olympus IX70 confocal laser-scanning microscope (Olympus, Tokyo, Japan).
Cm1900 cryostat
Manufactured by Leica
Sourced in Germany, United States, United Kingdom, Spain
The CM1900 cryostat is a laboratory instrument used for the preparation of frozen tissue samples for microscopic examination. It provides a controlled, low-temperature environment for sectioning and mounting biological specimens.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Mab377, by Merck Group (4 mentions)
A1r microscope, by Nikon (4 mentions)
Ab13970, by Abcam (4 mentions)
Tissue adhesive coated glass slides, by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Neg 50, by Thermo Fisher Scientific (3 mentions)
Eclipse ti microscope, by Nikon (3 mentions)
Prolong gold mounting media, by Thermo Fisher Scientific (3 mentions)
Lsm 880 confocal microscope, by Zeiss (3 mentions)
Permafluor, by Thermo Fisher Scientific (2 mentions)
Eclipse 80i microscope, by Adobe (2 mentions)
Vectashield mounting medium with 4 6 diamidino 2 phenylinidole dapi, by Vector Laboratories (2 mentions)
Ab15580, by Abcam (2 mentions)
Ab 16044, by Abcam (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Tissue tek, by Sakura Finetek (2 mentions)
Colorfrost plus slides, by Thermo Fisher Scientific (2 mentions)
Tissue freezing medium, by Leica (2 mentions)
156 protocols using cm1900 cryostat
1
Immunofluorescence Visualization of Photoreceptors
2
Postmortem Muscle Fiber Analysis
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Light microscopy of muscle samples at 24 h postmortem was conducted as described by Barbut et al. (2005) (link). All muscle sections (2.0×2.0×0.5 mm3) were cut along the direction of muscle fibers then placed in 10% formalin for 10 h to prefix the structure and then were dehydrated serially with 75%, 85%, 95%, and 100% ethanol followed by xylene. Samples were then embedded in paraffin, cut into 4 to 6 μm thick sections (Leica CM1900 Cryostat, Leica, Wetzlar, Germany; PowerTome XL, RMC-Boeckeler, Tucson, AZ, USA) and transferred onto a glass slide. Slides were dried, stained with hematoxylin-eosin, and observed using a light microscope (Axio Scope.A1, Carl Zeiss, Oberkochen, Germany) at 20×magnification. Pictures were captured using a computerized image analysis system (Image-Pro Plus v4.5, Media Cybernetics Inc., Silver Spring, MD, USA).
3
Epiblast Lineage Tracing in Embryos
4
Epiblast Lineage Tracing in Embryos
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At the pre-streak stage, Stage XII, embryos were injected with DiI and DiO (Molecular Probes) into cells of the lateral and medial region of the epiblast layer. Lateral region corresponds to the explant #2–3 described above, while medial regions correspond to explant # 6–7. Embryos were cultured at 37° C for 25–48 h in EC culture (Chapman et al., 2001 (link)), then fixed in 4% paraformaldehyde for 15 min before immunostaining. Embryos were mounted in gelatin and sectioned at 12 μm using a Leica CM1900 Cryostat. Sections were mounted with Permafluor (Thermo Scientific). Images were acquired on Nikon Eclipse 80i microscope and processed in Adobe Photoshop.
5
Immunohistochemical Analysis of Brain
6
NKCC1 and KCC2 Expression Mapping in Mouse Cortex
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The detection of NKCC1 and KCC2 mRNA by RNAscope kit (Advanced Cell Diagnostics) was done according to the supplier’s instructions, with minor modifications. P90 mice were terminally anesthetized by intraperitoneal injection of pentobarbital, followed by cardiac perfusion with ice-cold sterile PBS. Brains were immediately removed and frozen on dry ice. Coronal sections of 20 μm thickness were cut on a Leica CM1900 cryostat, mounted on glass slides (Super-FrostPlus; VWR International), and stored at −80 °C. Sections containing parietal and/or somatosensory cortex were chosen for analysis. Before hybridization, slices were postfixed in 4% paraformaldehyde (on ice) for 30 min. The specific NKCC1 and KCC2 in situ hybridization probes (Cat No. 311911-C2 and 311901, respectively) were provided by Advanced Cell Diagnostics. Slices were counterstained with 4,6-diamidino-2-phenylindole (DAPI, 1 μg/μl in PBS), mounted with ProLong Gold (Life Technologies), and stored at +4 °C until imaging.
7
Formalin-Fixed Embryo Sectioning
8
Quantifying Retinal ROS Production
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The eyecup was dehydrated for 30% sucrose and embedded in the optimal cutting temperature compound (tissue-Tek; Sakura, Torrance, CA, USA). The retinal specimen was cut vertically into sections using a Leica CM1900 cryostat (Leica, Wetzlar, Germany). The ROS production was evaluated by dihydroethylamine (DHE; BBoxiProbe, BB-470516) staining. The DHE fluorescence probes were diluted 2000 times with pure water to prepare for the dye probe working solution. Each section was incubated with DHE at 37°C for 30 minutes, and then rinsed with PBS for 5 minutes × 3 times. Retinal sections were photographed at 40x magnification using a fluorescence microscope and quantified using ImageJ software.
9
Muscle Fiber Type Identification
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Staining methods to determine muscle fiber type have been described previously [25] (link). Briefly, 8 µm thick frozen cross-section of each soleus muscle were sliced at −22°C using Leica CM1900 Cryostat (Leica, Nussloch, Germany). To distinguish between type I and IIa fibers in the soleus, myosin ATPase activity was measured following preincubation at pH 4.55 for 4 min. More than 300 fibers were counted in histochemical sections for quantitation of the fiber ratio.
10
Cryosection Imaging of Kidney Tissue
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Cryosections from frozen kidney tissues (5 μm) were prepared using a Leica CM1900 cryostat (Leica). As previously described [47 (link)], the sections were stained with dihydroethidium (DHE) solution for 30 min in the dark at 37 °C and then washed three times with phosphate-buffered saline (PBS). Finally, a fluorescence microscope (Olympus; Tokyo, Japan) was used to photograph the section.
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