Embryos were collected from the uterus of the pregnant mice under anesthesia. The heart of the embryo was visualized after removal of the skin and thoracic muscles. Then, transcardial perfusion of phosphate-buffered solution (PBS) at pH 7.4 and successive perfusion of 4% paraformaldehyde solution (PFA) in PBS at pH 7.4 were conducted through an infusion into the left ventricle of the heart. After the perfusion, the brain was postfixed for 24 h in 4% PFA solution. Then, the brain was sequentially immersed in 10%, 20%, and 30% sucrose solution in PBS for three consecutive days. The fixed brain was embedded in O.C.T compound (Sakura Finetek Japan Co. Ltd., Tokyo, Japan) with hexane (cat no. 082–00426, Wako Pure Chemical Industries Ltd., Japan) and dry ice. Neonatal mice at P0.5 were anesthetized by cooling the body temperature with indirect contact with ice. Mice were then transcardially perfused with PBS, followed by perfusion with 4% PFA through infusion into the left ventricles. The skin of the head was separated from the body and the brain was collected. Thereafter, the fixed brain was postfixed for 24 h in 4% PFA and immersed for three consecutive days in 10%, 20%, and 30% sucrose solution. The brain was then embedded in O.C.T compound (Sakura Finetek Japan Co. Ltd., Tokyo, Japan).
Oct compound
Manufactured by Sakura Finetek
Sourced in Japan, United States, Germany, Netherlands, Switzerland, United Kingdom, France
The OCT compound is a specialized laboratory equipment designed for optical coherence tomography (OCT) analysis. It enables high-resolution, non-invasive imaging of biological samples by utilizing low-coherence interferometry. The core function of the OCT compound is to capture detailed, cross-sectional images of various materials and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (25 mentions)
Lsm 700, by Zeiss (19 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (19 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (16 mentions)
Cryostat, by Leica camera (15 mentions)
Lsm 710, by Zeiss (15 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (15 mentions)
Mas coated glass slide, by Matsunami (13 mentions)
Paraformaldehyde (pfa), by Fujifilm (13 mentions)
Lsm 510, by Zeiss (13 mentions)
Cm3050, by Leica Microsystems (12 mentions)
Paraformaldehyde (pfa), by Merck Group (12 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (12 mentions)
Bz 9000, by Keyence (11 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (11 mentions)
Bx51 microscope, by Olympus (10 mentions)
Cm3050, by Leica camera (10 mentions)
Bovine serum albumin (bsa), by Merck Group (10 mentions)
Cryostat, by Leica Microsystems (9 mentions)
Fv3000, by Olympus (9 mentions)
1 063 protocols using oct compound
1
Perfusion and Embedding of Mouse Brains
2
Lung Tissue Fixation and Staining
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The lungs were perfused with PBS followed by fixation by intratracheal instillation of phosphate-buffered paraformaldehyde or OCT compound (Sakura Finetek, Tokyo, Japan) for embedding in paraffin or OCT compound, respectively. They were sectioned and subjected to hematoxylin and eosin (H&E) staining, periodic acid-Schiff (PAS) staining and immunostaining as described [18] (link), [19] (link).
3
Senescence Detection in Murine Lungs
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The right middle lung lobe of additional animals was inflated with Tissue-Tek O.C.T. Compound (#4583, Sakura Finetek USA, Inc., Torrance, CA, United States) through the trachea, embedded in Tissue-Tek O.C.T. Compound and frozen in a bath of isopentane pre-cooled in liquid nitrogen. Five μm cryosections were cut using a cryostat (HM525 NX cryostat, Thermo Fisher Scientific, Waltham, MA, United States), and tissue was processed with the Senescence Detection Kit (#K320-250, BioVision Inc., Milpitas, CA, United States), following the manufacturer’s protocol. Briefly, sections were fixed 10 min with the Fixative Solution of the kit at RT, and then incubated overnight at 37°C with the Staining Solution Mix containing 1 mg/ml X-gal. Development of the blue color linked with the beta-galactosidase activity was followed under the microscope. N = 3 animals/genotype.
4
Visualization of IL-26 signaling in cancer cells
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E0771 and HCC70 cells (1 × 105) were incubated in RPMI 1640 containing 10% FCS on Lab-Tek chamber slide (ThermoFisher Scientific) for 24 h. After incubation, cells were stimulated with Alexa Fluor 488-labeled IL-26 (30 ng/ml) in the presence or absence of recombinant human and mouse EphA3-Ig for 1 h. HCC70 cells were stimulated with IL-26 (30 ng/ml) in the presence or absence of Gefitinib (40 μM), signal inhibitors, and neutralizing antibodies for 24 h. Subcutaneous tumor samples obtained from mice were fixed in 4% paraformaldehyde (ThermoFisher Scientific), embedded in OCT compound (Tissue-Tek, Sakura Finetek, Tokyo, Japan). Cells and slides were immunostained with each antibody and observed utilizing Zeiss inverted microscope and Apotome.2. program (Carl Zeiss, Oberkochen, Germany). Fluorescence intensity was quantitated using Image-J software (NIH). Images were captured using objectives of ×100–×400.
5
Histological Analysis of Aortic Aneurysms
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Cryosections of both aneurysms and healthy aortas were used for histological analysis. Aortas were fixed in buffered formalin, embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) after being washed in deionized (DI) water and sectioned per standard procedures. Five-micrometer sections were mounted on positively charged glass slides. Slides were placed in 100% pre-cooled acetone (Fisher Science Education, Nazerath, PA) for 10 minutes to adhere the tissues to the slides. Subsequently, the slides were rinsed with tap water for 3 minutes to remove the OCT compound for further staining. Slides were then stained with Verhoeff-van Gieson (VVG) to visualize the elastin damage in different samples.
6
Tumor Antigen Visualization via Fluorescence Microscopy
7
Histological Evaluation of Enucleated Eyes
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For histological evaluation, enucleated eyes were fixed in 4% paraformaldehyde overnight and then embedded in paraffin or OCT compound (Sakura-Finetek, MA, USA). Fixed tissues were cut into 4 μm-thick sections and mounted on microscope slides. The sections were used for H&E staining or immunostaining according to the standard protocol. Fluorescence-conjugated isolectin B4 (#I21411, Thermo Fisher) and antibodies against Ninj1 (custom-made antibodies prepared by Abfrontier, Seoul, Korea) [4 (link)], F4/80 (#MCA497G, AbD Sertotec, Oxford, UK), β-galactosidase (#AB1211, Millipore, MA, USA), and cleaved caspase-3 (#9661, Cell Signaling Technology, MA, USA) were used. Nuclei were counterstained with Hoechst 33342 (Life Technologies, Carlsbad, CA, USA). Images were obtained using an Axiovert M200 microscope (Zeiss, Iena, Germany). The random fields in each section were calculated using ImageJ software and the relative expression level of each protein was quantified according to integrated optical density from three independent experiments. P values were calculated using the log-rank test.
8
Neuroanatomical Tracing of Corticospinal Projections
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The animals were deeply anesthetized and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. Then, the brain and cervical spinal cord were dissected out and post-fixed in the same fixative solution overnight at 4°C. The samples were cryoprotected in 30% sucrose in PBS and then embedded in an optimal cutting temperature (OCT) compound (Sakura Finetek). The brain and cervical spinal cord were cut serially into 50- and 20-μm thick coronal sections, respectively. To plot the lesion area in the sensorimotor cortex, coronal brain sections were stained with Nissl staining (0.1% cresyl violet). For BDA staining, sections of the cervical spinal cord were incubated in 0.3% Triton X-100 in PBS for 4 h, followed by Alexa Fluor 568-conjugated streptavidin (1:400, Invitrogen) in 0.1% Tween 20 in PBS for 2 h at room temperature as described previously (Ueno et al., 2012 (link), 2018 (link)).
9
Quantitative Histopathological Analysis of Hepatic Lipid Accumulation
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For the histopathological analysis, we used Oil Red O staining using the Oil Red O stain kit (Polysciences Inc., Warrington, PA, USA) based on previous studies. Liver samples were excised and embedded in an OCT compound (Sakura Finetek Japan Co., Osaka, Japan), and serially sectioned at 10 μm. The slides were immersed in 10% formalin for about 10 min, propylene glycol solution for 3 min, Oil Red O solution for 10 min, and 85% propylene glycol solution for 4 min in sequence. After rinsing with deionized water for 2 min, the specimens were stained with hematoxylin solution for 40 s. They were washed with tap water again for 3 min, and kept in deionized water, and finally sealed with Aqua-Poly/Mount (Polysciences Inc., Warrington, PA, USA). All tissue specimens were photographed using a fluorescence microscope (BZ-8100; Keyence Co., Osaka, Japan). The size of the lipid droplets was measured using Image J to quantify the fat accumulation in the liver.
10
Tracking Therapeutic Extracellular Vesicles
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iMSC-ER or pan PPAR-iMSC-EVs were stained with 5 μM CellTracker™ Orange CMTMR tetramethylrhodamine (Thermo Fisher Scientific) for 30 min at 37 °C. The stained EVs were subsequently isolated by ultracentrifugation at 100,000×g for 80 min, after which the pellet was washed with PBS and ultracentrifuged again (Beckman Coulter). After redissolution in EV-free PBS, 100 μL (400 μg) of iMSC-EVs or pan PPAR-iMSC-EVs were injected into the tail vain 8 h after cisplatin administration. Mice were euthanized by CO2 asphyxiation 72 h after cisplatin injection. The kidneys were then embedded in an OCT compound (Sakura Finetek, Torrance, CA, USA) and sectioned at 10 μm using a cryotome (Leica, Wetzlar, Germany). Sections were then stained with phalloidin-iFluor 488 (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Finally, the sections were mounted with a mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed under a confocal microscope (Leica TCS SP8 STED; Leica Camera AG, Wetzlar, Germany).
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