The whole right hind-limb was resected during [18F]-FDG biodistribution. Tissue was incubated at 4 °C for 1 h in optimal cutting temperature media (Fisher Healthcare, Houston, TX, USA). The limb was then transferred to a fresh optimal cutting temperature-filled mold and flash frozen on dry ice. The limb was rapidly sectioned using a custom modified CM1800 cryostat (Leica, Wetzler, Germany) at 10 μm. Standard sensitivity phosphor-screens (PerkinElmer) were exposed to the section in order to visualize [18F]-FDG uptake as needed for digital autoradiography and read using a Cyclone Phosphorimager (PerkinElmer).
Cm1800 cryostat
Manufactured by Leica
Sourced in Germany, United States
The CM1800 cryostat is a compact, versatile instrument designed for sectioning frozen tissue samples. It features a refrigeration system that maintains a stable low-temperature environment, enabling precise control of the cutting process. The CM1800 cryostat is a reliable tool for researchers and laboratories requiring high-quality frozen sections for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Optimal cutting temperature media, by Thermo Fisher Scientific (1 mentions)
Cyclone phosphor imager, by PerkinElmer (1 mentions)
Sucrose, by Merck Group (1 mentions)
Fluoview software, by Olympus (1 mentions)
Permount solution, by Thermo Fisher Scientific (1 mentions)
Tissue tek oct compound, by Thermo Fisher Scientific (1 mentions)
Superfrost plus microscope slides, by Leica (1 mentions)
A1r si point scanning confocal microscope, by Nikon (1 mentions)
Fluoroshield, by Merck Group (1 mentions)
Cy3 streptavidin, by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Biotinylated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Dm5000 b epifluorescent microscope, by Leica (1 mentions)
Rabbit anti mouse iba1, by Fujifilm (1 mentions)
Superfrost plus, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Cytation 3 cell imaging multi mode reader, by Agilent Technologies (1 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (1 mentions)
22 protocols using cm1800 cryostat
1
Biodistribution of [18F]-FDG in Tissue
2
In Vivo Tracking of Adoptively Transferred T Cells
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Naïve CD3+ T cells were treated with CS (100 µM) or vehicle as described above and stained with CMFDA or CMRA for 30 min at 37°C, respectively. The labeled cells were mixed at a 1:1 ratio in 200 µl of PBS and adoptively transferred to recipient mice i.v. Spleen and popliteal LNs were harvested at 2 h post-injection and fixed in 4% paraformaldehyde in PBS at 4°C overnight. On the next day, the samples were washed and incubated in PBS with 30% sucrose (w/v) (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C. The samples were then embedded in Tissue-Tek® O.C.T. Compound (Thermo Fisher Scientific) and frozen using 2-methylbutane, cooled with liquid nitrogen. 10 µm sections were cut using a Leica CM1800 cryostat. For immunostaining, tissue sections were blocked for 2 h in 10% normal goat serum at RT. The sections were incubated with fluorescently conjugated anti-B220 Ab at RT for 30 min in 10% goat normal serum. The samples were washed three times to remove unbound Ab and mounted in Permount solution (Thermo Fisher Scientific). Images were acquired with a confocal microscope and analyzed with FluoView software (Olympus). The epidermal/dermal depths and CD3ζ-3,3'-diaminobenzidine (DAB)+ cells were measured on 12 slides from 6 mice using EVOS FL Auto 2 software (Invitrogen).
3
Cryopreservation of Zebrafish Eyes
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Zebrafish eyes were fixed in 4% paraformaldehyde for 24 h, at 4 °C, and cryoprotected overnight in 25% (OCT) compound medium in 25% sucrose solution. Eyes were oriented in a plastic mold in 100% OCT, cooled down and stored at -80 °C for sectioning [27 (link)]. Then 10 μm serial sections were prepared at −25 °C, using a Leica CM1800 cryostat.
4
Cryoprotection and Sectioning of Ovaries
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After washing the ovaries in phosphate-buffered saline (PBS), they were fixed with 4%
paraformaldehyde in PBS overnight at 4°C. The ovaries were cryoprotected by incubation in
30% sucrose in PBS overnight at 4°C. Next, they were embedded in optimal cutting
temperature compound (Sakura Seiki, Tokyo, Japan) and frozen in liquid nitrogen. Sections
(7 µm thick) were cut with a CM1800 cryostat (Leica, Nussloch, Germany),
and the sections were placed on CREST coated glass slides (Matsunami Glass, Osaka, Japan)
and dried.
paraformaldehyde in PBS overnight at 4°C. The ovaries were cryoprotected by incubation in
30% sucrose in PBS overnight at 4°C. Next, they were embedded in optimal cutting
temperature compound (Sakura Seiki, Tokyo, Japan) and frozen in liquid nitrogen. Sections
(7 µm thick) were cut with a CM1800 cryostat (Leica, Nussloch, Germany),
and the sections were placed on CREST coated glass slides (Matsunami Glass, Osaka, Japan)
and dried.
5
Whole-Brain Tissue Preparation for Imaging
6
Immunofluorescence of Mouse Embryo Sections
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Dissected mouse embryos were fixed overnight in 4% paraformaldehyde in PBS followed by 3 washes in PBS. Fixed embryos were embedded in 7.5% gelatin/15% sucrose and sectioned into 10 μm slices using a Leica CM1800 cryostat. Sections were washed 2 × 5 min in Milli-Q and 1 × 5 min in PBS, permeabilised in 0.1% Triton X-100 in PBS for 20 min, and blocked in 5% normal donkey serum (in 0.05% Triton X-100 in PBS) for 1 h. Sections were incubated in a primary antibody overnight at 4°C followed by 4 × 10 min washes in 0.05% Triton X-100 in PBS, then incubated in a secondary antibody (1 : 1000) for 1 h at room temperature. Finally, sections were washed 3 × 5 min in 0.05% Triton X-100 in PBS and 2 × 5 min in PBS. Images were acquired using a Nikon A1R Si Point Scanning Confocal microscope, and fluorescent analysis was performed using SIESTA [41 (link)].
7
Preparation of Mouse Tissue Samples
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Mice were anesthetized with ketamine/xylazine and transcardially perfused with PBS followed by perfusion with 4% PFA. Mice were then decapitated and the skin was removed from the whole heads using forceps and scissors to separate the skin from the muscle and ear canal. The whole heads were fixed in 4% PFA overnight. The whole heads were then decalcified in 14% EDTA for 7 days followed by cryoprotection in 30% sucrose for 3 days (Supplementary Fig. 1 ). The EDTA was replaced with fresh 14% EDTA each day. For whole spinal column preparations, the skin was removed and the spinal column separated from the rib cage. The spinal columns were then fixed in 4% PFA overnight, decalcified in 14% EDTA for 7 days followed by cryoprotection in 30% sucrose for 3 days. The decalcified mouse heads and spinal columns were then embedded in Tissue-Tek OCT Compound, frozen in dry ice, then stored at −80 °C. Sixty-micrometer-thick frozen sections were obtained on a Leica CM1800 cryostat, mounted on Superfrost Plus microscope slides, and stored at −80 °C.
8
Cryogenic Preservation of Oocytes
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Oocytes were pipetted out of the well plate and placed in a disposable base mold (Fisher Scientific). The excess solution was then pipetted out of the base mold to prevent ice crystals from forming in the optimal cutting temperature (OCT) cryotome mounting media. To prevent the oocyte membranes from rupturing due to direct exposure to air, the oocyte was immediately covered in OCT compound completely filling up the mold. The mold was then placed in a −80 °C freezer for 30 minutes to ensure complete freezing and then stored at −20 °C. Oocytes in OCT compound blocks were sectioned at 10 µm using a CM1800 Cryostat (Leica, Wetzlar, Germany). Sections were taken from both the vegetal and animal poles. Sections were then placed on slides and stored at −20 °C. Prior to imaging, the mounting medium Fluoroshield (Sigma-Aldrich, St. Louis, MO, USA) was applied to the sections and coverslips were placed on each slide.
9
Quantitative Analysis of Mitochondrial SOD2 in Liver Tissue
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Liver tissue sections (5 microns) were obtained from snap-frozen biopsies using a Leica CM1800 Cryostat. Staining of mitochondrial marker SOD2 was performed using rabbit anti-SOD2 (NB100-1969, Novus Biologicals) at 1:200, followed by incubation with a biotinylated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories Inc.) and Cy3-streptavidin (Jackson ImmunoResearch Lab. Inc.). Tissues were counterstained for DNA using DAPI (Roche Diagnostics Corp.). Images were captured and processed as described for immunofluorescence microscopy. Automated image analysis for the quantification of subcellular SOD2 protein content was performed using the CellProfiler software (http://www.cellprofiler.org ) (Carpenter et al, 2006 (link)) and a self-made analysis pipeline. Briefly, channel intensity was calculated for at least 10 individual images per treatment for the DAPI (blue) channel and for the Cy3 (red) channel. Specific signal intensity for SOD2 staining was calculated by normalizing the Cy3 channel intensity to DAPI intensity. Data were normalized to the WT signal (set as 1) and are depicted as an average + s.d. Unpaired two-tailed t-test was used to calculate P values between the treatments (**P < 0.01).
10
Immunohistochemical Analysis of Microglia and Astrocytes
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Mice were sacrificed by CO2 asphyxiation and transcardially perfused with ice-cold PBS (pH 7.4) and 4% paraformaldehyde (PFA). Brains were collected and post-fixed in 4% PFA for 24 h, cryoprotected in 30% sucrose for 24 h, frozen using dry ice-cooled isopentane (− 78 °C), sectioned coronally at 30 μm on a Leica CM1800 cryostat, and stored in cryoprotectant (30% ethylene glycol, 30% polyethylene glycol, 40% 0.2 M phosphate buffer) for immunolabeling. Next, sections (Bregma - 1.5 mm) were washed in PBS, blocked (5% normal donkey serum, 0.1% Triton X-100, 1% bovine serum albumin in PBS) for 1 h, and incubated with rabbit anti-mouse Iba1 (1:1000; Wako Chemicals) or rabbit anti-mouse GFAP antibody (1:500; Abcam) overnight at 4 °C. Next, sections were washed in PBS and incubated with a fluorochrome-conjugated secondary antibody (Alexa Fluor 594 or Alexa Fluor 488). Sections were mounted on slides and cover-slipped with Fluoromount-G (Invitrogen). Slides were then imaged using a Leica DM5000 B epifluorescent microscope at 20X magnification and captured using a Leica DFC300 FX camera and imaging software. For each animal, 2–4 images were quantified and averaged, and these values were used to calculate the mean and standard error for each experimental group.
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