1-, 7-, 14-, and 30-days after SE, rats were deeply anesthetized with pentobarbital (75-100 mg/kg, i.p.) and transcardially perfused with PBS (100 mL) followed by 4% paraformaldehyde (200 mL). Brains were removed and post-fixed in 4% paraformaldehyde overnight at 4°C, then transferred to a solution of 30% sucrose in PBS for 72 hours, and frozen with dry ice before storage at -80°C until sectioning. A 1-in-5 series of sections containing the amygdala was cut at 40 μm on a sliding microtome (Leica Microsystems SM2000R). One series of sections was mounted on slides (Superfrost Plus, Daigger, Vernon Hils, IL) for Nissl staining with cresyl violet. Two adjacent series of sections were mounted on slides for Fluro-Jade-C staining or were stored at -20°C in a cryoprotectant solution for GAD-67 immunohistochemistry (Figueiredo et al., 2011b (link)).
Sm2000r
Manufactured by Leica Microsystems
Sourced in Germany
The Leica SM2000R is a high-performance stereomicroscope designed for a variety of laboratory applications. It features a binocular observation tube, providing a comfortable and ergonomic viewing experience. The microscope offers a range of magnification levels, allowing users to closely examine samples with precision. Its core function is to provide clear and detailed observation of specimens under examination.
Automatically generated - may contain errors
Lab products found in correlation
Tp1020, by Leica Microsystems (1 mentions)
Eg1160, by Leica Microsystems (1 mentions)
Adhesive slide mas coated glass slides, by Matsunami (1 mentions)
3 aminopropyltriethoxysilane, by Shin-Etsu Chemical (1 mentions)
Cm1950, by Leica Microsystems (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Image pro insight software, by Media Cybernetics (1 mentions)
Mouse monoclonal anti neun, by Merck Group (1 mentions)
Vector elite abc kit, by Vector Laboratories (1 mentions)
Somnopentyl, by Kyoritsu Seiyaku (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Rm2245, by Leica Microsystems (1 mentions)
Maxymum recovery, by Corning (1 mentions)
Eclipse e800, by Nikon (1 mentions)
Paraformaldehyde (pfa), by Leica Microsystems (1 mentions)
Sleepaway, by Zoetis (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Donkey anti mouse 488, by Jackson ImmunoResearch (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Donkey anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
21 protocols using sm2000r
1
Rat Brain Tissue Fixation and Sectioning Protocol
2
Thymic Tissue Sectioning and Preservation
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Eight-week-old male mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg body weight) and perfused from the left ventricle with 0.1 M phosphate-buffered saline (PBS), pH 7.4 for 10 min and then with 4% paraformaldehyde in PBS for 10 min. Thymic tissues collected from the mice were fixed with 4% paraformaldehyde in PBS for 24 h at room temperature (RT) and then embedded in paraffin using a tissue processor (TP1020; Leica Microsystems, Wetzlar, Germany) under the following conditions: 70% (v/v) ethanol; 80% ethanol; 90% ethanol; three washes with 100% ethanol; three washes with xylene; and embedding twice in paraffin wax for 1 h each time. The FFPE blocks prepared using a paraffin-embedding device (EG1160; Leica Microsystems) were sectioned on a sliding microtome (SM2000R; Leica Microsystems) equipped with disposable blades (S35; Feather Safety Razor, Osaka, Japan). For LMD, 5-μm-thick tissue sections were mounted on poly-l -lysine-coated PEN- and PPS-membrane glass slides (Leica Microsystems). For immuno- and lectin histochemistry, 2-μm-thick sections were placed onto Matsunami Adhesive Slide (MAS)-coated glass slides (Matsunami, Osaka, Japan). The sections were dried overnight at 42 °C and stored at RT until use.
3
Organ Weight and Tissue Sectioning
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We measured the organ weights of the thymus, spleen and liver after trimming. We cut the ear auricles vertically by using razors (FEATHER Safety Razor Co., Osaka, Japan), then embedded one
cross section in Tissue-Tek® OCT compound and fresh-froze it (Sakura Finetek, Tokyo, Japan). The other cross sections of the ears and small blocks of other organs
were fixed in 4% paraformaldehyde in phosphate buffer for 24 hr at 4°C, dehydrated through a graded series of ethanol followed by xylene, and embedded in paraffin. Finally,
4-µm-sections were cut by a cryostat (CM1950; Leica Microsystems, Wetzlar, Germany, for frozen-sections) or a sliding microtome (SM2000R; Leica Microsystems, for
paraffin-sections), and mounted on a glass slide precoated with 0.2% 3-aminopropyltriethoxysilane (Shin-Etsu Chemical Co., Tokyo, Japan).
cross section in Tissue-Tek® OCT compound and fresh-froze it (Sakura Finetek, Tokyo, Japan). The other cross sections of the ears and small blocks of other organs
were fixed in 4% paraformaldehyde in phosphate buffer for 24 hr at 4°C, dehydrated through a graded series of ethanol followed by xylene, and embedded in paraffin. Finally,
4-µm-sections were cut by a cryostat (CM1950; Leica Microsystems, Wetzlar, Germany, for frozen-sections) or a sliding microtome (SM2000R; Leica Microsystems, for
paraffin-sections), and mounted on a glass slide precoated with 0.2% 3-aminopropyltriethoxysilane (Shin-Etsu Chemical Co., Tokyo, Japan).
4
Kidney Tissue Histopathological Evaluation
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Kidney tissue was collected at the conclusion of the experiments, cut into small blocks (3 mm thickness), and fixed by immersion in 4% buffered formalin. The blocks were embedded in paraffin, and 2 to 4 mm tissue slides were prepared using a microtome (SM 2000R, Leica Instruments, Nußloch, Germany). Hematoxylin eosin staining was conducted adherent to in-house standards and used to assess morphological integrity of the parenchyma.
Assessment was carried out in an anonymous fashion in 10 randomly chosen, nonoverlapping fields (×400 magnification), using a 5-point scale for tubular dilatation, vacuolization, glomerular damage, and tubular shedding as described previously17 (link): 0 = no damage; 1 = lesions affecting <10% of the field; 2 = 10% to 25%; 3 = 25%–50%; 4 = 50% to 75%; and 5 > 75%. In each individual kidney, the mean value from the 4 respective parameters was calculated and taken as individual mean histological injury score.
Assessment was carried out in an anonymous fashion in 10 randomly chosen, nonoverlapping fields (×400 magnification), using a 5-point scale for tubular dilatation, vacuolization, glomerular damage, and tubular shedding as described previously17 (link): 0 = no damage; 1 = lesions affecting <10% of the field; 2 = 10% to 25%; 3 = 25%–50%; 4 = 50% to 75%; and 5 > 75%. In each individual kidney, the mean value from the 4 respective parameters was calculated and taken as individual mean histological injury score.
5
Immunohistochemical Thalamic Lesion Analysis
6
Perfusion and Sectioning Protocol for Amygdalar Analysis
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Seven (7) days after CCI, animals were deeply anesthetized using nembutal (75–100 mg/kg, i.p.) and transcardially perfused with phosphate buffered saline (PBS, 100 ml) followed by 4% paraformaldehyde (250 ml). Brains were removed and post-fixed in 4% paraformaldehyde overnight at 4°C, then transferred to a solution of 30% sucrose in PBS for 72 hours, and frozen with dry ice before storage at −80°C until sectioning. Sectioning was performed as previously described [33] (link), [34] (link). A 1-in-5 series of sections containing the rostrocaudal extent of the amygdala was cut at 40 µm on a sliding microtome (Leica Microsystems SM2000R). A 1-in-5 series of free-floating sections was collected from the cryoprotectant solution, and washed three times for 5 min each. Slices were mounted on a slide, air-dried overnight and processed for Nissl staining with cresyl violet while the adjacent series of sections were mounted on slides for Fluoro-Jade C (FJC) staining.
7
Histological Analysis of Mandibular Tissues
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The mandibular tissues were removed, decalcified with decalcifying liquid (K-CX; Falma, Tokyo, Japan) for 3 days, fixed in 10% paraformaldehyde, and paraffin-embedded. Next, sections (5 μm thick) were cut longitudinally using a microtome (SM2000R; Leica Microsystems, Wetzlar, Germany), and stained with hematoxylin and eosin. For immunohistochemical analysis, after deparaffinization, the endogenous peroxidases of the sections were blocked with 3% hydrogen peroxide (H2O2). The sections were then immunostained with anti-cathepsin K antibody (NCL-CATH-K, Novocastra, Newcastle, UK) for 60 min in room temperature. After primary antibody staining, the sections were washed with tris-buffered saline and incubated with a secondary antibody (EnVisionTM Reagent; K4000, Dako, Glostrup, Denmark) for 30 min in room temperature. The sections were developed using diamino benzidine substrate.
8
Perfusion and Sectioning Protocol for Mouse Brain Analysis
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Mice were killed at 1 h, 4, 8 and 12 weeks post-intraventricular injection, 1, 2, 3 and 14 months post-cortical injection, or 3 months post-intravenous injection. All mice were subjected to intra-cardiac perfusion with PBS followed by perfusion with 4% PFA under deep anesthesia with 1% ketamine (30 mg/kg) and xylazine hydrochloride (4 mg/kg). Brains were collected, post-fixed in 4% PFA overnight and subsequently stored in 20% sucrose in PBS for cryoprotection. For the intra-ventricular injection protocol, the 8 and 12 weeks time points as well as all time points from the other experiments, were cut into 25-μm-thick coronal brain sections using a sliding microtome (Leica Microsystems, ON, CA, Cat# SM 2000R), serially collected in anti-freeze solutions and stored at − 20 °C until use. For intra-ventricular injections, the 1 h and 4 week time points, were cut into 12-μm-thick coronal brain sections using a cryostat (NX 70, Thermo Scientific) and mounted sections were stored at − 20 °C until use.
9
Immunohistochemical Analysis of Pancreas
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Mice were deeply anesthetized using pentobarbital sodium (50 mg/kg body weight, intraperitoneal injection, Somnopentyl, Kyoritsu Seiyaku, Tokyo, Japan) and perfused transcardially with saline followed by 4% paraformaldehyde (PFA; 26126-25, Nacali Tesque Inc., Kyoto, Japan) in phosphate-buffered saline (PBS). The pancreas was dissected, cut into small pieces, and post-fixed in 4% PFA overnight at 4°C. Post-fixed specimens were dehydrated and then embedded in a paraffin block. The paraffin block was sectioned at 5 µm thickness on a microtome (SM 2000R, Leica Microsystems, Wetzler, Germany). The sections were used after deparaffinization and rehydration.
10
Postnatal Developmental Analysis in Pups
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The male pups in each group were analyzed at 24 h after birth for Nissl staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), immuhohistochemistry and electron microscopy, and at around postnatal day 40 (P40) for Nissl staining. To induce hypothermia as anesthesia, they were immersed up to the neck in crushed ice and water and transcardially perfused with 4% paraformaldehyde with 0.1 M phosphate buffer (PB; pH 7.2; Fujifilm Wako, Osaka, Japan or Nacalai Tesque, Kyoto, Japan). The brains were dissected out and postfixed in the same fixative overnight at 4°C and embedded in paraffin. Coronal sections at 4-μm thick were cut with a microtome (SM2000R or RM2245; Leica Microsystems, Nussloch, Germany).
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