Liver tissue was fixed in 10% formalin and then embedded in Tissue-Tek optimal cutting temperature compound. Frozen liver tissues sectioned to 30 μm thick using Cryostat (Leica CM 3000) were subjected to hsSRS imaging analysis as previously described (53 (link), 62 (link), 63 (link)). Briefly, the spectral focusing–based hsSRS system was used, with a dual-output femtosecond laser (InSight DeepSee; Spectra-Physics) providing pump (800 nm) and Stokes (1040 nm) pulses at an 80 MHz repetition rate. An electro-optical modulator (EO-AM-R-C2; Thorlabs) was used to modulate the Stokes laser at a resonant frequency of 10.5 MHz. The time delay line controlled by a motorized stage was employed, and the microscope (BX51; Olympus) equipped with a water objective (UPLSAPO 60XW; Olympus) was used for laser scanning and imaging. The pump beam was detected by a photodiode (S3994-01; Hamamatsu) with two installed short-pass filters (ET980SP; Chroma), and the SRS signals were acquired by a lock-in amplifier (HF2 LI; Zurich Instruments). The laser power of pump and Stokes beams were set to 50 and 70 mW (measured before galvanometer), respectively.
Cm 3000
Manufactured by Leica camera
Sourced in Germany
The CM 3000 is a cryostat, a laboratory instrument used for sectioning frozen biological samples. It is designed to maintain a precise, low-temperature environment for preparing samples for microscopic examination and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (12 mentions)
Mab377, by Merck Group (2 mentions)
Zen software, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Insight deepsee, by Spectra-Physics (1 mentions)
Uplsapo60xw, by Olympus (1 mentions)
Hf2li, by Zurich Instruments (1 mentions)
Ax70 light microscope, by Olympus (1 mentions)
Anti ctip2, by Abcam (1 mentions)
Anti parvalbumin, by Merck Group (1 mentions)
Anti neun, by Merck Group (1 mentions)
Fluoromyelin green, by Thermo Fisher Scientific (1 mentions)
Anti olig2, by Abcam (1 mentions)
Alexa 488 or 594 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Nanoject, by Drummond (1 mentions)
Sm2000r, by Leica camera (1 mentions)
Avidin biotin peroxidase complex abc, by Vector Laboratories (1 mentions)
28 protocols using cm 3000
1
High-Speed Hyperspectral SRS Imaging of Liver Tissue
2
Immunohistochemical Analysis of Mouse and Rat Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemical analysis, mouse and rat tissue was prepared as described thereafter. The heads from E15.5 embryos, newborn pups and 2 week-old mice were fixed in 4% paraformaldehyde (PFA) at 4°C overnight and then transferred into 30% sucrose/0.1 M phosphate buffer solution until sectioning on cryostat apparatus (10–14 μm thickness; Leica CM3000). Sections were mounted in series on Superfrost glass slides and stored at −80°C until immunohistochemistry was performed. Six week-old mice and two and six week-old rats were sacrificed by transcardial perfusion with saline for 5–10 minutes, followed by 4% PFA for 10 minutes. Brains were fixed and cryopreserved as described above until sectioning on a microtome apparatus (30 μm thickness sections, Microm Zeiss). Seven series of coronal sections were cut throughout the brain. Free-floating sections were preserved in anti-freeze solution until immunohistochemistry was performed.
3
Muscle Fiber Cross-Sectional Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cross-sectional areas from muscle fibers (types 1, 2A, and 2B) were calculated and analyzed. TA muscles were sectioned in a Leica CM3000 cryostat (8 μm) and sections were treated using the metachromatic dye-ATPase method (mATPase) according to Ogilvie and Feeback (1990) [29 (link)]. Slides with dried frozen sections were placed in a preincubation solution composed of distilled water (475 mL), dehydrated potassium acetate (2.45 g), and calcium chloride (1.30 g) at pH 4.5 for 2 minutes; slides were subsequently rinsed in Tris buffer. Slides were then incubated for 25 minutes in an incubation solution composed of distilled water (30 mL), potassium chloride (185 mg), ATP, disodium salt (76 mg), dehydrated calcium chloride, (0.18 M = 5 mL), and Sigma 221 buffer (1.5 M 2-amino-2-methyll-propanol) (3.35 mL) at pH 9.4. Slides were rinsed by dipping in three changes of 1% calcium chloride solution, stained with 1% aqueous toluidine blue for 10 seconds, and immediately rinsed in distilled water. Images of stained muscle sections were acquired on an Olympus AX70 light microscope (Olympus Melville, NY). Cross-sectional areas from types 1, 2A, and 2B fibers, from five slides per animal (300 to 400 fibers/animal), were measured using the Image J software (NIH).
4
Immunohistochemistry of Adult Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adult mouse brains (approximately 8 weeks of age) were harvested, fixed in 4% paraformaldehyde (PFA) overnight, cryoprotected in 30% sucrose and frozen at − 80 °C before further processing. 20 μm-thickness frozen coronal sections were cut using a microtome cryostat system (Leica CM3000). Free floating sections were initially blocked in 5% fetal bovine serum, 1% Triton X-100, 0.5% Tween 20 and 1% skim milk in 0.1 M PBS for 2 h at room temperature to reduce non-specific binding. This was followed by incubation with primary antibodies overnight at 4 °C and secondary antibodies for 2 h in blocking solution at room temperature. The primary antibodies used in this study include: anti-NeuN (1:200, Millipore MAB377, Etobicoke, Canada), anti-Parvalbumin (1:200, Sigma-Aldrich P3088, Oakville, Canada), anti-CDP (M-222) (Cux1) (1:200, Santa Cruz Biotechnology sc-13,024, Dallas, TX, USA), anti-Ctip2 (1:200, Abcam ab28448, Cambridge, MS, USA), anti-Olig2 (1:200, Abcam ab109186), anti-Iba1 (1:500, Wako 019–19,741, Richmond, VA, USA) and anti-GFAP (1:200, Dako Z0334, Glostrup, Denmark). Alexa 488- or 594-conjugated secondary antibodies (1:200; Thermo Fisher Scientific, Burlington, Canada) in blocking solution were used for detection of primary antibodies. Staining of myelin and nuclei was performed with Fluoromyelin green and DAPI (Thermo Fisher Scientific) respectively.
5
Hippocampal Morphological Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The obtained tissue sections (six brains/group) were collected on glass slides, deparaffinized, stained with 1% cresyl violet solution by Nissl staining for morphological study of the hippocampus, and then examined under a light electric microscope (CM 3000; Leica, Bensheim, Germany).
6
Perfusion and Brain Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After MRI scanning sessions, the animals were anesthetized with a Ketamine/Xelazine solution and then perfused intracardially and exsanguinated with a peristaltic pump conveying 0.9% NaCl followed by 4% PFA diluted in PBS. After this fixation phase, animals were decapitated, and the brain removed and post-fixed in 4% PFA overnight. Thereafter, the brains were placed for 48 h in a 20% PBS-sucrose solution and then frozen in isopenthane at −43°C to be subsequently sliced (30 or 50 μm thick) using a cryostat (Leica CM 3000).
7
Perfusion Fixation of Mouse Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) after the behavior test on the 15th day and rapidly perfusion fixed with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Then, the brains were removed and stored at −80°C until use. The frozen brains were cut into 18 μm coronal sections using a cryostat microtome (CM3000; Leica, Germany). Frozen sections of the striatum and substantia nigra were used for immunohistochemistry and autoradiography experiments.
8
Tracing Thalamo-Cortical Connectivity in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
P18 hevin KO and WT littermate mice were deeply anesthetized with intraperitoneal injection of ketamine (150 mg/kg)/xylazine (15 mg/kg). Using Nanoject (Drummond, PA), mice were stereotactically injected by 50 nl of EF-1a promoter-driven Flex-AAV-GFP within the dLGN [AP: −2.0; ML: 2.0; DV: 2.3 from brain surface]. To visualize specific neurons in dLGN that are directly connected with visual cortex, 100 nl of rabies virus glycoprotein-coated Lenti-FuGB2-Cre (synapsin promoter) (Kato et al., 2011 (link)) was infected into the V1 region of visual cortex [AP: −3.5; ML: 2.5; DV: 0.3 from brain surface]. 2 weeks after infection, brains were removed, postfixed overnight at 4°C, and then cryo-protected with 30% sucrose in TBS. Brains were cut into 50 μm coronal sections by cryostat (Leica CM 3000). Sections were counterstained with DAPI (Sigma). After washing three times, the sections were coverslipped with FluorSave (CalBioChem, Merck, Germany) aqueous mounting medium. For the axonal fiber tracing, images were taken by tile scan imaging using LSM 710 confocal microscope (Zeiss, Germany) with a 10× objective under control of Zen software (Zeiss).
9
Histological Analysis of Tibialis Anterior Muscle
10
Retrograde Labeling of Motor Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two months after treatment, six animals from each group were anesthetized with an intraperitoneal injection of a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg). After general anesthesia, 40 μl of saturated DiI (1, 1-dioctadecyl-3, 3, 3, 3–tetramethylindocarbocyanin perchlorat) from Molecular Probes (Leiden, Netherlands; cat. No, D-282) in DMSO was injected at four points in the left gastrocnemius muscle [20 (link)]. Ten days later, the rats were deeply anesthetized with a double dose of ketamine (200 mg/kg) and xylazine (20 mg/kg) and perfused via a cardiac vessel with 0.9% heparin and with a fixative containing 4% paraformaldehyde and 1.25% glutaraldehyde in a 0.1 M/L sodium phosphate buffer (pH 7.2). Lumbar spinal segment L4-L6 was dissected out and placed in 30% sucrose overnight. Serial 50 μm-thick transverse sections of the spinal segment were made using a freezing microtome (leica cryostat, CM 3000). In each group, the labeled motor neurons were counted in the left anterior horn using fluorescent microscopy (Olympus Ax70).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!