Wild-type littermate (n=3) and 5XFAD (n=3) female mice at 6 months of age were anesthetized and perfused transcardially with PBS and 4% PFA in PBS and post-fixed in 4% PFA for 20 h. The brain slices were sectioned to a 30-μm thickness with a CM 1950 cryostat (Leica Microsystems GmbH, Nussloch, Germany). Free-floating sections of the frontal cortices, which contain plenty of capillaries, were pretreated with 70% formic acid for 20 min to retrieve the antigens and then incubated with the following primary antibodies: biotin-labeled 4G8 (1 : 700; Covance, Inc.), rabbit anti-P-gp (1 : 100; Santa Cruz Biotechnology, Inc.), Fluorescein Lycopersicon esculentum (Tomato) lectin (1 : 100; Vector Laboratories, Inc., Burlingame, CA, USA), rat anti-Lama2 (1 : 500; Abcam plc, Cambridge, MA, USA), and rabbit anti-Aqp4 (1 : 100; EMD Millipore Corporation) overnight at 4°C. Following extensive washes in PBS, the sections were incubated with secondary antibodies that were conjugated with Alexa Fluor dyes (1 : 500; Life Technologies Corporation) for 2 h at RT. Immunofluorescent images were taken with the aid of a confocal laser scanning microscope (FV10i-w; Olympus Corporation).
Cm1950 cryostat
Manufactured by Leica Microsystems
Sourced in Germany
The CM1950 is a cryostat designed for sectioning tissue samples at low temperatures. It features a motorized cutting system and a temperature range of -20°C to -50°C. The CM1950 is intended for use in histology, pathology, and research laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Fluorescein lycopersicon esculentum tomato lectin, by Vector Laboratories (1 mentions)
Fv10i w, by Olympus (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Rabbit anti aqp4, by Merck Group (1 mentions)
2 methylbutane, by Merck Group (1 mentions)
Anti atp5a, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions)
Blocking one histo, by Nacalai Tesque (1 mentions)
Fv1000d, by Olympus (1 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
Biotinylated anti mouse igg antibody, by Vector Laboratories (1 mentions)
Permount solution, by Thermo Fisher Scientific (1 mentions)
Hematoxylin and eosin solution, by Merck Group (1 mentions)
Fsc22 frozen section media, by Leica Microsystems (1 mentions)
Imsol mount, by Immunologic (1 mentions)
Roti histofix, by Carl Roth (1 mentions)
Osteoimagetm mineralization assay, by Lonza (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Biorevo bz 9000 microscope, by Keyence (1 mentions)
14 protocols using cm1950 cryostat
1
Brain Vasculature and Barrier Imaging in 5XFAD Mice
2
Tissue Preparation for Confocal Microscopy and qPCR
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For confocal studies, mice were deeply anesthetized by intraperitoneal (ip) injection of a mixture of ketamine and xylazine and transcardially perfused with 25–30 mL of saline solution for 5 min, followed by 10 min with 4% PFA (Sigma), pH 7.4, in 0.1 M phosphate buffer (PB, Sigma). Brains were obtained and post fixed with 4% PFA for 18–20 h at 4 °C, rinsed in 0.1 M PB and placed in 15% glucose at 4 °C until they sank followed by immersion in 30% sucrose in PB at 4 °C for 72 h. Finally, brains were embedded in tissue freezing medium (Tissue-Tek O.C.TTM, Sakura), by submerging brains in increasing concentrations of OCT, snap frozen in dry-ice-cooled 2-methylbutane (Sigma) and stored at −80 °C. Coronal sections (30 μm) were obtained with a CM1950 cryostat (Leica Microsystems) and stored at −20 °C until use.
To perform qPCR on brain samples, brains were dissected and tumours were extracted and snap-freeze. Samples were liquid-nitrogen-cooled pestle and mortar fragmented for subsequent analysis.
To perform qPCR on brain samples, brains were dissected and tumours were extracted and snap-freeze. Samples were liquid-nitrogen-cooled pestle and mortar fragmented for subsequent analysis.
3
Confocal Imaging of Mouse Hippocampus
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For confocal preparations, mice were deeply anesthetized by intraperitoneal (ip) injection of a mixture of ketamine and xylazine and transcardially perfused with 25–30 mL of saline solution for 5 min, followed by 10 min incubation with 4% paraformaldehyde (PFA, Sigma), pH 7.4, in 0.1 M phosphate buffer (PB, Sigma). After perfusion with the fixative, brains were dissected out and post fixed with 4% PFA for 18–20 h at 4°C. After fixation, brains were rinsed in 0.1 M PB and placed in 15% sucrose at 4°C until they sank, and then in 30% sucrose in PB at 4°C for 72 h. Finally, brains were embedded in tissue freezing medium (Tissue-Tek O.C.TTM, Sakura), by submerging them in increasing concentrations of OCT, frozen immediately in dry-ice-cooled 2-methylbutane (Sigma), and stored at −80°C. Coronal sections (30 μm) were cut using a CM1950 cryostat (Leica Microsystems). Brain sections were collected sequentially in 10 slides. Six sections per slide generate antero-posterior reconstructions of the hippocampus conformed by 1 section every 300 μm of hippocampal structure. Slides were stored at −20 °C until use.
4
Quantification of Mitochondrial Morphology
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Frozen liver tissues were sectioned (control, homozygous sample number, n = 3) into 10-µm sections using a CM 1950 cryostat (Leica Microsystems, Wetzlar, Germany) and fixed with 4% paraformaldehyde (Nacalai Tesque, Tokyo, Japan) for 20 min. The sections were washed (thrice, 5 min each) with 1 × PBS and incubated with a blocking reagent (Blocking One Histo, Nacalai Tesque) for 20 min at 25 °C. After washing with 1 × PBS (thrice, 5 min each), the sections were incubated overnight with anti-ATP5a (1:100; Abcam, Cambridge, UK) at 4 °C, followed by washing with 1 × PBS (thrice, 5 min each) and incubating with anti-rabbit IgG Alexa 488 (1:1000; Abcam) for 2 h at 25 °C. The sections were washed with 1 × PBS again (thrice, 5 min each) and incubated with a drop of 4′,6-diamidino-2-phenylindole (DAPI) for 20 min at 25 °C, followed by confocal laser microscopy (FV1000 D; Olympus, Tokyo, Japan). The obtained data were analyzed using image analysis software (Photoshop; Adobe, Mountain View, CA, USA), and the total mitochondrial area per cell was calculated.
5
Ex Vivo Ligated Ileal Loop Assay
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The ex vivo ligated ileal loop assay was performed as previously described (Primard et al., 2010 (link); Ma et al., 2014 (link)) with some modifications. Briefly, female BALB/c mice (6–8 weeks old) were fasted overnight and anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg animal weight). Subsequently, 2 cm of the ileal loop containing a PP was ligated and injected with PLGA-coumarin-6 NPs or UEA-1/PLGA-coumarin-6 NPs diluted in PBS (10 mg/mL). After 1-h incubation, mice were euthanized by cervical dislocation. The ligated ileal loop was excised and washed with PBS, followed by fixation with 4% paraformaldehyde for 1 h at room temperature. Frozen sections (10 μm) of PP were obtained using a CM1950 cryostat (Leica Microsystems, Wetzlar, Germany). Tissue samples captured on Superfrost plus microscope slides (Thermo Scientific, USA) were washed with PBS three times to remove any residual optimal cutting temperature compound. Samples were then blocked with PBS containing 5% FBS and stained with DAPI. CLSM images were recorded using an Ultra View VOX CLSM instrument (PerkinElmer, USA).
6
IgG Immunohistochemistry in Aged Mouse Brains
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Immunohistochemistry for IgG staining was performed as described previously with some modifications. Briefly, 9‐ to 10‐month‐old mice were anesthetized and perfused sequentially with PBS and 4% PFA in PBS, and the brain samples were fixed again in 4% PFA for 20 h. The slices were sectioned with a CM 1950 cryostat (Leica Microsystems GmbH, Nussloch, Germany). For IgG staining, the mouse brain sections were washed twice with ice‐cold PBS and pretreated with 3% H2O2 for 30 min at room temperature. After washing three times, the sections were incubated overnight with biotinylated anti‐mouse IgG antibody (Vector, Burlingame, CA, USA) and then visualized with 3,3‐diaminobenzidine tetrahydrochloride (DAB) by the avidin–biotin–peroxidase complex (ABC) method. Tissues were mounted, air‐dried, dehydrated by alcohol, and immersed in xylene. They were cover slipped with Permount solution (Thermo Fisher Scientific) and imaged using a fluorescence microscope (IX71; Olympus Corporation, Tokyo, Japan). The acquired images (prefrontal cortex regions) were analyzed by Image J software. IgG intensity was measured under the same threshold line, and the average values were determined and quantified.
7
Tissue Cryosectioning and Staining
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The excised tissues were immediately fixed with 4% paraformaldehyde solution for 24 h, washed with PBS, and immersed in 30% sucrose solution for 2 days to cryoprotect the tissues. After embedding in FSC22 Frozen Section Media (Leica Microsystem), tissues were frozen and sliced (10 -20 μm thick) using a cryostat (CM1950 cryostat, Leica Microsystems). The tissue sections were finally stained with hematoxylin and eosin solutions (Sigma-Aldrich), and images were taken using a digital microscope (Leica DVM6).
8
Retinal Dark Adaptation Histology
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Mice were kept in a dark room overnight and their eyes were dissected in the dark with infrared goggles (NV-G1, Japan Medical services, Tokyo, Japan). Dissected eyes were placed in DMEM. One eye from each pair was kept in the dark and the other was illuminated. After 30 min of incubation, all pairs of eyes were fixed with 4% PFA in 0.1 M phosphate buffer (PB) (pH 7.2) for 6 hr during the fixation, dark-adapted eyes were shielded from light. The eyes were then cryoprotected with 10% and then 20% sucrose/PBS and embedded in optimal cutting temperature compound (SAKURA Finetek, Tokyo, Japan). Sections were cut in the plane parallel to the optic nerve with a cryostat (CM1950 Cryostat, Leica Microsystems, Wetzlar, Germany). For immunostaining, sections within 300 μm from the meridian plane were used. Each pair of eyes from the same mouse (where one is dark-adapted and the other is light-adapted) were put on the same glass slide and stained under identical conditions and stored at −80 °C until use.
9
Cryosectioning and Mineralization Assay
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The printed constructs were washed in PBS and fixated for 15 min at room temperature using 4% formalin (ROTI®Histofix; Roth, Karlsruhe, Germany). After washing in PBS, the printed constructs were embedded in an optimal cutting temperature (OCT) compound (Sakura Finetek, Alphen aan den Rijn, Netherlands) and incubated at 37 °C for 35 min. Samples were shock-frozen in liquid nitrogen and stored at −80 °C until further use. Following this, 10 µm cryosections were produced using the CM1950 cryostat (Leica Microsystems, Wetzlar, Germany). To assess the mineralization of the printed matrix, samples were stained using the OsteoImageTM Mineralization Assay (Lonza, Basel, Switzerland) according to the manufacturer’s protocol. Briefly, sections were permeabilized with acetone for 10 min at −20 °C, washed two times with PBS and once with a wash buffer, incubated with the staining reagent for 30 min at room temperature, washed three times with a wash buffer, and mounted using ImsolMount (ImmunoLogic, Duiven, Netherlands). Images were taken using the BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan).
10
MALDI Tissue Imaging Sample Preparation
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Tissue sections of 4 µm thickness were cut using CM1950 cryostat (Leica Microsystems, Wetzlar, Germany) and mounted onto indium-tin-oxide (ITO) coated glass slides (Bruker Daltonik GmbH, Bremen, Germany), which were previously covered with 1:1 poly-L-lysine (Sigma-Aldrich; Taufkirchen, Germany) and 0.1% Nonidet P-40 (Sigma-Aldrich; Taufkirchen, Germany). For deparaffinization, sections were incubated at 70 °C for 1 h and washed twice in xylene for 8 min. Prior MALDI matrix application, tissue sections were air-dried on a heating plate at 35 °C for 1 min and scanned using a flatbed scanner in order to acquire digital tissue images for co-registration purposes. Subsequently, the samples were covered with 10 mg/mL 9aminoacridine matrix (Sigma-Aldrich) in 70% methanol (purity ≥99.9%), using a SunCollect sprayer (Sunchrom, Friedrichsdorf, Germany) according to Ly et al. [33] . In detail, the following preferences were applied for the automatic sprayer: vial distance of 0.50 mm for the X direction and 2.00 mm for the Y direction, 20 mm Zposition and offset of the spray head, and medium X/Y speed. The matrix was deposited in eight layers using variable increasing spray rates. Following spray rates were used: Layer 1: 10 µl/min, layer 2: 20 µl/min, layer 3: 30 µl/ min, layer 4-8: 40 µl/min. At all, the whole procedure is resulting in a total amount of 0.16 mg/cm 2 9-aminoacridine.
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