Seven days after the surgery, six mice in each group were deeply anesthetized with an overdose of 3.5% chloral hydrate (0.1 mL/10 g, i.p), and transcardially perfused with 0.9% NaCl solution to rinse out the blood, followed by perfusion with 250 mL of 4% formalin (4°C) to fix the muscle tissue. The muscle tissue was postfixed in 4% paraformaldehyde and subsequently cut into 6 µm coronal sections by a cryostat (Leica CM1900, Germany) for HE staining, acetylcholinesterase assay, and TUNEL and LC3-II staining by fluorescent immunohistochemical technology. Another eight muscle tissue samples in each group were immediately frozen using liquid nitrogen and stored at −70°C until further analysis. Serial transverse 6-µm-thick sections were cut by a cryostat (Leica CM1900). Ipsilateral muscles (0.1 g per muscle) in each group were also weighed for Western blotting analysis of LC3II/LC3I, BCL2/BAX, and mTOR signaling protein, and biochemical determination of SOD and MDA.
Cm1900
Manufactured by Leica
Sourced in Germany, United States, United Kingdom, Japan, China
The Leica CM1900 is a cryostat, a device used for the preparation of frozen tissue sections for microscopic analysis. It provides a controlled, low-temperature environment to facilitate the cutting and mounting of tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (10 mentions)
Mvx10, by Olympus (9 mentions)
Paraformaldehyde (pfa), by Merck Group (9 mentions)
Tissue tek, by Sakura Finetek (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (9 mentions)
Image pro plus 7, by Olympus (8 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (7 mentions)
Dm4000b, by Leica (6 mentions)
Tcs sp2, by Leica (5 mentions)
Lsm 780, by Zeiss (5 mentions)
Tissue tek oct compound, by Sakura Finetek (5 mentions)
Eclipse 80i, by Nikon (5 mentions)
Superfrost plus, by Thermo Fisher Scientific (5 mentions)
Superfrost plus slides, by Avantor (4 mentions)
Tissue tek oct, by Sakura Finetek (4 mentions)
Fluorescence microscope, by Zeiss (4 mentions)
Oct compound, by Sakura Finetek (4 mentions)
Axio imager z2 microscope, by Zeiss (4 mentions)
Mounting medium, by Vector Laboratories (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
472 protocols using cm1900
1
Muscle Tissue Analysis after Surgery
2
Nasopharyngeal Carcinoma Tissue Collection
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As our previous study14 (link), NPC samples (256 cases) and non-tumor nasopharyngeal epithelium (NPE) (131 cases) were collected in Xiangya Hospital and the Second Xiangya Hospital (Changsha, PR China) during 2002 to 2004. All biopsies were immediately fixed in 4% buffered paraformaldehyde, routinely processed and embedded in paraffin, and then prepared the NPC tissue microarray. The clinicopathologic characteristics were listed in Table 1 . Among all of the NPC patients, we followed up 81 patients to do survival analysis. The time of following up was from 4 to 95 month, and average was 57 month.
For the mRNA expression study, another 36 NPC tissues and 15 NPE samples were obtained from patients in the Hunan Cancer Hospital/The Affiliated Cancer Hospital of Xiangya School of Medicine (Changsha, China) in 2013. All tissue samples were quickly frozen in liquid nitrogen and stored at -80°C until laser-capture micro-dissection (LCM). We used a LEICA CM 1900 (Leica, Solms, Germany) for frozen sections and the Leica AS LMD system (Leica) to obtain the pure tissues.
All of the individuals participating in this project signed the informed consent form and their clinicopathologic characteristics, such as name, sex, age, metastasis and clinical stages were recorded.
For the mRNA expression study, another 36 NPC tissues and 15 NPE samples were obtained from patients in the Hunan Cancer Hospital/The Affiliated Cancer Hospital of Xiangya School of Medicine (Changsha, China) in 2013. All tissue samples were quickly frozen in liquid nitrogen and stored at -80°C until laser-capture micro-dissection (LCM). We used a LEICA CM 1900 (Leica, Solms, Germany) for frozen sections and the Leica AS LMD system (Leica) to obtain the pure tissues.
All of the individuals participating in this project signed the informed consent form and their clinicopathologic characteristics, such as name, sex, age, metastasis and clinical stages were recorded.
3
Apoptosis Quantification in Spheroids
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On day 6, spheroids were fixed with methanol for 10 min in −20 °C, washed in PBS, embedded in Tissue-Tek OCT compound, and then stored at −80 °C until used for experiments. Frozen sections were obtained at 7 μm thickness using a Cryostat (Leica CM1900, Leica, Wetzlar, Germany) in −25 °C to −30 °C. The sections were washed and permeabilized in 0.2% Triton X-100 in PBS for 5 min at RT. The sections were then washed and stained using the TUNEL apoptosis detection kit. The extent of the DNA breaks in apoptotic cells was evaluated by a fluorescence microscope.
4
Quantifying Muscle Fiber Morphology
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Determination of the cross-sectional area (CSA) and the percentage of slow and fast muscle fibers can be found in our previous reports [44 (link),50 (link)]. In brief, the soleus muscle sections were prepared with a Leica CM 1900 cryostat (Leica, Braunschweig) at −20 °C. Sections were incubated with primary antibodies MyHC I(β) slow (1:100 Sigma, St. Louis, MO, USA), MyHC fast (1:60, DSMZ) for 1 h at 37 °C and secondary antibodies Alexa Fluor 546 (1:1000; Molecular Probes, Waltham, MA, USA) for 60 min in the dark at room temperature. The soleus muscle sections were photographed with a Leica Q500MC fluorescent microscope at magnification ×20. Image analysis was processed by the ImageJ 1.52a software. At least 150 fibers were analyzed in each muscle sample (n = 8) for myofiber CSA measures, and at least 10 cross-sections per sample were examined to determine the percentage of different muscle fiber types in the sample (n = 8).
5
Microscopic Analysis of Hongkong Kumquat Plastids
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Protoplasts from the calli were generated as previously described [69 ], then protoplast suspensions were dropped onto microscope slides to observe the plastid modes. Light microscopy of various orange tissues of Hongkong kumquats was performed using a frozen sectioning technique with a Leica CM1900 (Leica, Germany). An optical microscope (BX61, Olympus) equipped with a DP70 camera was used in tandem with a differential interference contrast (DIC) technique.
Transmission electron microscopy (TEM) analysis was performed according to Cao et al. [28 (link)]. Samples were prepared using a normal fixation process with 2.5% glutaraldehyde adjusted to pH 7.4, and a 0.1 M phosphate buffer with 2% OsO4. The preparations were dehydrated and embedded in epoxy resin and SPI-812, respectively. Ultrathin sections obtained with a Leica UC6 ultramicrotome were stained with uranyl acetate and subsequently with lead citrate. Image recording was performed with a HITACHI H-7650 transmission electron microscope at 80 KV and a Gatan 832 CCD camera.
Starch granule morphology was examined with a scanning electron microscope (SEM). The samples were mounted on studs, sputter coated with gold (Balzers, JFC-1600), and examined under a JSM-6390LV SEM (JEOL, Japan).
Transmission electron microscopy (TEM) analysis was performed according to Cao et al. [28 (link)]. Samples were prepared using a normal fixation process with 2.5% glutaraldehyde adjusted to pH 7.4, and a 0.1 M phosphate buffer with 2% OsO4. The preparations were dehydrated and embedded in epoxy resin and SPI-812, respectively. Ultrathin sections obtained with a Leica UC6 ultramicrotome were stained with uranyl acetate and subsequently with lead citrate. Image recording was performed with a HITACHI H-7650 transmission electron microscope at 80 KV and a Gatan 832 CCD camera.
Starch granule morphology was examined with a scanning electron microscope (SEM). The samples were mounted on studs, sputter coated with gold (Balzers, JFC-1600), and examined under a JSM-6390LV SEM (JEOL, Japan).
6
Histochemical Staining of Mouse Lungs
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Following histochemical staining, images of the desired regions were obtained using a stereo-fluorescence microscope and processed using the Olympus software package Image-ProPlus 7.0. Mouse lungs were treated by triformol, then treated with OCT mounting medium before sectioned at 10 µm using a cryostat microtome (Leica CM1900; Leica, Wetzlar, Germany). Images were obtained using an Olympus IX51 epi-fluorescent microscope (at × 200 and × 400) (Olympus, Tokyo, Japan) and analysed using the CW4000 FISH Olympus software.
7
Brain Tissue Preparation for Confocal Imaging
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Thirteen days after LV-eGFP or LV-B injection, the rats were killed and the brains were fixed by transcardial perfusion with 0.1 m PBS for 10 min after the rats were deeply anesthetized with sodium pentobarbital (60 mg kg−1, intraperitoneally). Brains were removed and kept in 25 ml 4% PFA for 1 h and then were washed in 0.1 m PBS and immersed in 15% and subsequently 30% sucrose solution for 2 days. The brains were blotted dry and snap-frozen for 10 s in isopentane on dry ice and stored at −80 °C until sectioning. Serial coronal 20-μm-thick sections were obtained using a cryostat (Leica CM 1900, Leica, Wetzlar, Germany). All the brain sections containing the hippocampus were collected and thaw-mounted on Super Frost microscope slides. The slides were then stained with DAPI (4',6-diamidino-2-phenylindole) and confocal images were acquired using a confocal microscope (Leica, TCS SP2).
8
Hippocampal Tissue Preparation for IHC
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Five weeks after the first injection of BrdU, and a few days after the end of behavioural analysis, all animals were killed and intracardially perfused (see Supplementary Figure 1A ). This time point corresponds to ~5 weeks of exposure to both Ex and EE. Brain tissue was obtained and stored at −80 °C for immunohistochemistry as previously described.43 (link) Serial coronal hippocampal sections from Bregma coordinates −1.34 to −2.54 mm were cut on a cryostat (Leica CM1900, Leica Biosystems Melbourne, Mount Waverly, VIC, Australia) at 40-μm thickness, were collected in a 1 in 6 series spaced 240 μm apart, and immediately placed in a cryopreserve solution of 0.1 m Phosphate buffer containing 25% v/v ethylene glycol (VWR International, Leuven, Belgium) and 25% v/v (Chem-Supply, Gilman, SA, Australia). Collected series were then stored at −20 °C until used for peroxidase immunohistochemistry.
9
Whole-mount embryo imaging and sectioning
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Following immunofluorescent staining, the whole-mount embryos were photographed by using a stereo-fluorescence microscope (Olympus MVX10; OLYMPUS, Tokyo, Japan) and processed with a Olympus software package Image-Pro Plus 7.0. The embryos were then sectioned into 15-μm thick slices by using a cryostat microtome (Leica CM1900; LEICA, Solms, Germany) and photographed by using an epi-fluorescent microscope (Olympus IX51, Leica DM 4000B) at a magnification of 200 × and 400 × . The images were analysed and processed by using a CW4000 FISH Olympus software package.
10
Immunofluorescence Staining of Muscle Tissue
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Muscle tissues were fixed with 4% paraformaldehyde in PBS, and then cryoprotected in 30% sucrose at 4 °C overnight. The tissue sections (10 μm) were performed on a cryostat (Leica CM 1900; Leica, Milton Keynes, UK). Ten percent normal goat serum was used for blocking of non-specific binding at 4 °C overnight. The tissue sections were incubated with the primary antibodies (see Supplementary Table S1 ) and then rinsed with PBS and they were incubated with fluorescence conjugated secondary antibodies. All secondary antibodies are also listed in Table S1 ; these antibodies were incubated for 1 hr in the dark. Nuclei were counterstained by 4′6-diamino-2-phenilindole (DAPI, Sigma-Aldrich) at room temperature for 30 secs. After washing PBS, sections were mounted and coverslipped on glass slides using Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and analyzed by confocal microscopy (Carl Zeiss 710, Carl Zeiss, Germany).
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