Hematoxylin and eosin (HE) staining was performed at BioPathology Institute. Co., Ltd. (Kunisaki, Oita, Japan). Mandibular gingiva samples were fixed with 10% formalin solution (Fujifilm Wako, Osaka, Japan). After fixation, the tissues were washed under running tap water, dehydrated in ascending grades of ethanol, and cleared in xylene. Paraffin-embedded tissue sections 6 μm in thickness were cut using a microtome and mounted on glass slides. Histology and photomicrography were performed by Biopathology (Kunisaki, Oita, Japan).
Formalin solution
Manufactured by Fujifilm
Sourced in Japan
Formalin solution is a clear, colorless liquid composed of formaldehyde and water. It is a commonly used chemical reagent in various laboratory and medical applications, serving as a fixative agent to preserve biological samples for analysis and examination.
Automatically generated - may contain errors
Lab products found in correlation
Glass slide, by Matsunami (2 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Signal enhancer hikari, by Nacalai Tesque (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Fischer 344 rats, by Japan SLC (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Mas coated slides, by Matsunami (1 mentions)
Inverted phase contrast microscope, by Olympus (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Axioskop 2 plus, by Zeiss (1 mentions)
Axiocam mrc camera, by Zeiss (1 mentions)
Paraffin, by Sakura Finetek (1 mentions)
Cm 3000, by Leica (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
N n dimethylformamide (dmf), by Fujifilm (1 mentions)
Fast red violet lb salt, by Merck Group (1 mentions)
Technovit 9100, by Kulzer (1 mentions)
Cryostat, by Leica (1 mentions)
Bz 9000, by Keyence (1 mentions)
3 3 diaminobenzidine (dab), by Fujifilm (1 mentions)
21 protocols using formalin solution
1
Histological Analysis of Mandibular Gingiva
2
Histological Analysis of Mouse Dorsal Skin
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Dorsal skin was excised and fixed with 20% formalin solution (Fujifilm Wako Pure Chemical Corporation). The skin samples were embedded in paraffin by a conventional method and stained with hematoxylin and eosin. Images were acquired using an Olympus virtual slide system VS110 (Olympus, Tokyo, Japan). The thickness of the epidermis was expressed as the mean values from five fields per mouse.
For immunohistochemistry, cryosections of mouse dorsal skin were fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Corporation) for 10 min, washed with 0.1% Triton X-100 in phosphate-buffered saline for 10 min, and incubated with Blocking One (Nacalai Tesque, Kyoto, Japan) for 30 min. Following overnight incubation at 4 °C with primary antibodies, the sections were incubated with Alexa Fluor-conjugated secondary antibodies at room temperature for 60 min in the dark and mounted in PLUS Antifade Mounting Medium (Vector Laboratories, Newark, CA, USA). Antibodies were diluted with Signal Enhancer HIKARI (Nacalai Tesque). Images were acquired using an LSM 700 confocal laser microscope (Carl Zeiss Co., Ltd., Oberkochen, Germany).
For immunohistochemistry, cryosections of mouse dorsal skin were fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Corporation) for 10 min, washed with 0.1% Triton X-100 in phosphate-buffered saline for 10 min, and incubated with Blocking One (Nacalai Tesque, Kyoto, Japan) for 30 min. Following overnight incubation at 4 °C with primary antibodies, the sections were incubated with Alexa Fluor-conjugated secondary antibodies at room temperature for 60 min in the dark and mounted in PLUS Antifade Mounting Medium (Vector Laboratories, Newark, CA, USA). Antibodies were diluted with Signal Enhancer HIKARI (Nacalai Tesque). Images were acquired using an LSM 700 confocal laser microscope (Carl Zeiss Co., Ltd., Oberkochen, Germany).
3
FGF-2 Nanoparticle Skin Delivery in Rats
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Inbred male Fischer 344 rats (Japan SLC, Inc., Shizuoka, Japan) weighing 170–190 g were used in all the experiments. All rats were maintained on a standard laboratory diet with water ad libitum. Rats were anesthetized by intraperitoneal injections of 0.15 mg/kg medetomidine, 2 mg/kg midazolam, and 5 mg/kg butorphanol tartrate. The dorsal skin hair was shaved and depilated using a depilatory cream. Intraperitoneal injections of 0.15 mg/kg atipamezole roused the rats from the anesthesia. FGF-2&D/P NPs, standalone FGF-2, or saline (control) was applied to the dorsal skin twice daily for 3 days. In this experiment, each solution (0.2 mL) was rubbed into the skin, and then 0.1 mL of solution was in contact with the skin for 20 minutes or more to wake up from the anesthesia. On the third day, the rats were killed and their skin samples were taken.
Skin specimens were fixed in 10% formalin solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan), which were embedded in paraffin and sectioned at 4 μm. The sections were mounted onto glass slides and stained with FGF-2 antibody, which was performed at Kyodo Byori Inc. (Hyogo, Japan). All animal experiments were approved and carried out following the approval and guidelines for animal experimentation set by the National Defense Medical College, Saitama, Japan.
Skin specimens were fixed in 10% formalin solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan), which were embedded in paraffin and sectioned at 4 μm. The sections were mounted onto glass slides and stained with FGF-2 antibody, which was performed at Kyodo Byori Inc. (Hyogo, Japan). All animal experiments were approved and carried out following the approval and guidelines for animal experimentation set by the National Defense Medical College, Saitama, Japan.
4
Sirius Red Staining of Tissue Sections
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Frozen and paraffin tissue sections were used for Sirius red staining. After fixation of frozen tissue sections using 10% formalin solution (Wako), it was replaced with Milli-Q water (frozen tissue section). The slides were deparaffinized thrice for 10 min in xylene and dehydrated with 100%, 95%, 80%, 70% and 50% ethanol solutions. Then, it was replaced with Milli-Q water (paraffin tissue section). The slides were immersed in 0.03% Sirius red/saturated picric acid solution (1% Sirius red solution saturated with aqueous solution picric acid) for 30 min and rinsed under Milli-Q water. Afterward, these slides were immersed in 50%, 70%, 80%, 95% and 100% ethanol solutions and then in xylene for 3 min. Such steps were repeated thrice. Finally, Mount-Quick (Daichi Sangyo Co., Ltd.) was dropped, and the sample was covered with a glass slide (Matsunami Glass).
5
Histological Analysis of Mouse Liver
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Mice were anesthetized with 1–4% isoflurane with vaporization. The transcardial perfusion with saline in the anesthetized mice was performed, and then mice were euthanized prior to removal of the livers. The isolated livers were fixed in 10% formalin solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 12 h at 4°C, dehydrated in graded ethanol and xylene, and embedded in paraffin blocks for microtome slicing into 5 µm thick sections. The sections were placed on MAS-coated slides (Matsunami Glass Ind., Ltd., Osaka, Japan), dried, and used for subsequent studies. Sections were then used for conventional hematoxylin and eosin staining and Elastica van Gieson staining, using a kit according to the manufacturer's instructions (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Stained sections were photographed using an upright microscope (model ECLIPSE 80i; Nikon Corporation, Tokyo, Japan) with a digital camera. Quantitative analysis of hepatocyte hypertrophy was performed by imaging cytometric analysis (NIS-Elements D version 3.2; Nikon Corporation). The number of hepatocytes was counted using templates of regular squares, each of which enclosed an area of 200×200 µm from a total of 12 randomly-selected fields per group.
6
Hematoxylin and Eosin Staining Protocol
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For HE staining, we used the cryostat tissue sections (sections 1–4), which were first fixed with 10% formalin solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), followed by incubation with Milli-Q water. Next, glass slides were deparaffinized thrice for 10 min in xylene and dehydrated with 100%, 95%, 80%, 70%, and 50% ethanol solutions. Then, it was replaced with Milli-Q water (paraffin tissue section). The slides were immersed in Carrazi’s hematoxylin (Muto Chemistry) for 10 min and rinsed with running tap water to remove excess stain from the slide. Afterward, the slides were immersed in eosin (Muto Chemistry) for 10 min and rinsed with Milli-Q water. Next, these slides were immersed in 50%, 70%, 80%, 95% and 100% ethanol solutions and then in xylene for 3 min. Such steps were repeated thrice. Thereafter, Mount-Quick (Daichi Sangyo Co., Ltd.) was dropped, and the sample was covered with a glass slide (Matsunami Glass).
7
Colony-Forming Unit Assay for ASCs
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Aging ASCs and DFAT cells (1 × 103 cells/mL) were separately plated in 10-cm2 dishes (Nunc) and cultured in GM for 7 days. Then, cells were fixed with 10% formalin solution (Wako Pure Chemical Industries, Ltd, Japan) and stained with 0.1% toluidine Blue (Muto Pure Chemical, Japan) for 5 min. The aggregated colonies containing 50 or more cells and having a diameter exceeding 2 mm were counted under a phase contrast inverted microscope (Olympus Optical Co. Ltd, Tokyo, Japan). The CFU (%) was calculated as follow: number of colonies per plate/number of cells seeded × 100.
8
ATLL Tumor Histopathology and Apoptosis Analysis
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Tumor specimens were collected from the control group and the 30 mg/kg AUY922-treated group, fixed in formalin solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan), dehydrated through graded ethanol series (Japan Alcohol Trading Co., Ltd., Tokyo, Japan) and embedded in paraffin (Sakura Finetek Japan Co., Ltd., Tokyo, Japan). The paraffin-embedded specimens of ATLL tumors were stained with hematoxylin and eosin (H&E; Merck Millipore, Darmstadt, Germany). Cells were examined under a light microscope (Axioskop 2 Plus) with an Achroplan ×40/0.65 lens (both from Carl Zeiss AG, Oberkochen, Germany). Images were captured with an AxioCam MRc camera and AxioVision 4.7 software (Carl Zeiss AG). Analysis of DNA fragmentation by TUNEL assay was performed using a commercial kit (Roche Applied Science, Penzberg, Germany).
9
Quantifying Adipogenesis in 3T3-L1 Cells
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The 3T3-L1 cells were fixed with 10% (v/v) formalin solution (Wako Pure Chemical Industries, Osaka, Japan) two days after fET and the change to maturation medium. The fixed cells were incubated with 18 mg/ml Oil Red O in a 60% (v/v) 2-propanol solution for 20 min at room temperature and the lipid droplets in cells were observed by microscopy. Then, Oil Red O dissolved in the lipid droplets was extracted with 100% 2-propanol, and its relative concentration was determined by measuring absorbance at 540 nm.
10
MERS-CoV Neutralization Assay with Dromedary Sera
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For the neutralization assay, sera obtained from dromedaries as described above were used along with mice immunized with an EMS isolate (wild type) (Iwata-Yoshikawa et al., 2019 (link)). The neutralization assay for MERS-CoV was performed using Vero/TMPRSS2 cells as described (Shirato et al., 2015 (link)) with minor modifications. Serially diluted sera were mixed with 50–100 PFU of targeted viruses in 10% TPB-DMEM and incubated at room temperature for 45 min. The mixture was applied to Vero/TMPRSS2 cells in a 96-well plate, incubated for 24 h at 37°C, and then fixed with 10% formalin solution (Wako Pure Chemical Industries) under ultraviolet light. The fixed cells were then stained with crystal violet, and the number of cell fusions was counted under a microscope. Non-serum samples were used as a negative control. The serum dilution that showed 80% neutralization relative to the negative control was considered the antibody titer. Specimens showing neutralization at more than 20-fold dilution relative to the negative control were considered positive for MERS-CoV infection. A dot plot graph was drawn using KaleidaGraph ver. 4.5 (Synergy Software, Mt. Penn, PA, USA).
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