Animals were deeply anesthetized with ketamine/xylazine mixture and perfused transcardially with 0.1M phosphate buffer. Brains were dissected and divided sagittally. One half was immediately placed on dry ice and stored at − 80°C for biochemical studies while the other half was fixed in 4% paraformaldehyde at 4°C for 48h, and cryoprotected in 30% sucrose in 0.1M phosphate buffer at 4 °C until saturated. Fixed hemispheres were sectioned at 40 μm sagittally through the entire hemisphere using a sledge microtome (SM200R; Leica Biosystems). Free-floating sections were stored at − 20°C for immunostaining. Eyeballs from perfused mice were enucleated and fixed in 4% PFA overnight at 4 degrees. Following fixation, the cornea and the lens were removed to form an eyecup. The eyecup was transitioned through a 10%, 20%, and 30% sucrose gradient. Eyes were embedded in Tissue Plus O.C.T. Compound (Fisher HealthCare, 4585). Eyes were sectioned with a cryostat at 10 um thickness and mounted on Superfrost slides (Fisher Scientific, Pittsburgh, PA).
Sm200r
Manufactured by Leica
Sourced in Germany
The SM200R is a high-precision lab equipment designed for specialized applications. It features advanced optics and sensors to provide accurate measurements and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Oil red o, by Merck Group (2 mentions)
Tissue plus o c t compound, by Thermo Fisher Scientific (1 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Rabbit alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Oil red, by Merck Group (1 mentions)
2 methylbutane, by Merck Group (1 mentions)
5 bromo 2 deoxyuridine brdu, by Merck Group (1 mentions)
Ketamine, by Merck Group (1 mentions)
S8ap0, by Leica (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Goat anti 5 ht, by Immunostar (1 mentions)
17 protocols using sm200r
1
Transcardial Perfusion and Brain-Eye Tissue Processing
2
Immunofluorescent Labeling of Mouse Brain
3
Histological Preparation of Decalcified Knee Joints
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The backward knee joints specimens were kept in 10% v/v formalin solution for 24 h, washed by tap water for half an hour, and then the specimens were decalcified in the chelating agent disodium EDTA solution 4% w/v (Drury and Wallington, 1980 ) [30 ]. Decalcification lasted for about 4 weeks, during which the solution was renewed every 2 days until the tissues had softened. The decalcified knee joints were cleaved longitudinally in a sagittal plane along the central portion, and specimens were processed to form paraffin blocks. The fixed specimen was dehydrated after washing in ascending grades of ethyl alcohol for one hour for each as follows: 50%, 60%, 70%, 80%, 90%, and two times in 100% v/v (absolute alcohol). Then, the specimens were cleared with xylene and then for 15 min for each impregnated and embedded in paraffin at 60 °C. The blocks were cut into sections 5 microns thick by using a rotatory microtome (Leica SM 200R), were placed on numbered glass slides, and dried overnight in an oven at 37 °C. After this, the slides were immersed in two changes of xylene for 15 min for each and then were placed for 1 min in each of four changes of absolute alcohol. Consequently, the slides were placed in descending grades of alcohol from 90% and 70% to 50% v/v and then were immersed in distilled water. The sections were subjected to the following stains:
4
Histological Verification of Neuronal Recordings
5
Optogenetic Activation of Hippocampal CA1
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Following in vivo optogenetic experiments, mice were deeply anesthetized intraperitoneally with sodium pentobarbital (MTC Pharmaceuticals), perfused transcardially first with 0.1M PBS (PBS) and next with 4% paraformaldehyde in 0.1M PBS (PFA). Brains were isolated, postfixed in 4% PFA for 24 h and cryoprotected in 30% sucrose. Coronal brain sections (50 μm thick) were obtained using freezing microtome (SM200R; Leica) and mounted in Pro-Long Gold (Invitrogen). Sections were examined using an epifluorescence microscope (Eclipse E600; Nikon) and images were acquired with the Simple PCI software. Immunofluorescence was used to enhance GFP fluorescence associated with Arch. For these mice, brain sections were obtained as described above, permeabilized with 0.5% Triton X-100 in PBS for 15 min, and unspecific binding was blocked with 10% normal goat serum in 0.1% Triton X-100/PBS for 1 h. Rabbit polyclonal GFP (1/200) antibodies were incubated for 48 h at 4°C. Sections were subsequently incubated at room temperature with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1/500; 90 min), mounted in Pro-Long Gold, and imaged as described above. For behavioral experiments, data were excluded if mice did not show virus expression restricted to CA1, and if optic fibers placement was outside the CA1 hippocampus.
6
Histological Analysis of Liver Lipid Depots
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Liver samples were analysed for lipid and fat depots by Oil red O staining. Frozen samples were cut into 30 µm-thick sections using a sliding microtome (Leica SM200R, Wetzlar, Germany) and fixed with 10% formal calcium. Sections were washed with distilled water and rinsed with 60% isopropanol. Then, sections were stained with freshly prepared Oil red O (Sigma, St Louis, MO) working solution for 20 minutes (Oil red O stock stain: 0.5% of Oil red O in isopropanol; Oil red working solution: 30 ml of the stock stain and 20 ml of distilled water). Sections were rinsed with 60% isopropanol, counterstained with Mayer’s hematoxylin, rinsed with tap water and mounted in aqueous media.
7
Chimpanzee Brain Preservation and Sectioning
8
Rat Cartilage Histomorphometry and Ultrastructure
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Five male rats of the groups N, U, R, NN and RR were euthanized in a carbon dioxide (CO2) chamber. The CPs were dissected, fixed in Bouin’s solution for 8 h and decalcified17 for 30 days. After washing in distilled water, the tissues were dehydrated through an ascending series of alcohols (70 to 100%), diaphanized in xylene and paraffin-embedded. Semi-serial sections of 5 μm, in the sagittal plane, were obtained from the CPs using a microtome (Leica SM 200R, São Carlos, SP, Brazil), mounted on slides and placed in an oven for deparaffinization. The sections were stained with azocarmine23 to identify and measure cartilage layers thickness (TCPC) and the area of CP cartilage, as well as to quantify the number of cells of the chondroblastic layer. For the analysis and quantification of the cartilage matrix (CM) of the CP, the safranin-O method was used. To evaluate the collagen component, the picro-sirius red method was used. Samples were processed for immunohistochemistry to observe the sensitivity of the IR in CP cartilage and a structural analysis was performed by scanning electron microscopy (SEM) of the CP cartilage.
9
Microdialysis and Brain Sectioning Procedure
10
Cysteamine Treatment on Hippocampal Slices
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Hippocampal slices were obtained as above and transferred in oxygenated ACSF containing cysteamine (200 µM) or ACSF alone, for 1 h at room temperature. Slices were then used for electrophysiological whole cell recording (as described below) or fixed overnight at 4 °C with 4% PFA, rinsed with PB 0.1 M, cryoprotected in 30% sucrose/PB 0.1 M and re-sectioned (50 µm) using a freezing microtome (Leica SM200R, Germany) for immunofluorescence.
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