The Ti/cell sheets/HA complexes were then implanted subcutaneously into the dorsal surface of 6-week-old immunocompromised mice (Animal Center of the Fourth Military Medical University, Xi’an, People’s Republic of China). The mice were maintained in pathogen-free conditions at 26°C, at 70% relative humidity, and under a 12-hour light/dark cycle. All animal procedures used in this study were approved by the research ethics committee of The Fourth Military Medical University. There were four animals in each sample group. At 8 weeks after implantation, the mice were euthanized, and the implanted samples were harvested, fixed in 4% neutral formaldehyde (Sigma-Aldrich Corp), embedded in resin, and sectioned. The sections were stained with the Van Gieson staining, and hard-tissue histological analyses were performed using light microscopy.
Neutral formaldehyde
Manufactured by Merck Group
Sourced in United States
Neutral formaldehyde is a laboratory reagent used for the preservation and fixation of biological samples. It functions as a cross-linking agent, stabilizing and preserving the structural integrity of cells, tissues, and organisms. Neutral formaldehyde is commonly used in various applications within the life sciences, such as histology, cytology, and microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel coated chamber, by BD (1 mentions)
Te2000 u, by Nikon (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Avantor (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Oil red o, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Sevoflurane, by Merck Group (1 mentions)
Vacutainer, by BD (1 mentions)
8 protocols using neutral formaldehyde
1
Subcutaneous Implantation of Ti/cell/HA Constructs
2
Pesticide and Probiotic Effects on Rat Testes
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The study was carried out with 72 Wistar Albino male rats, weighing between 200–300 g, taken from the Experimental Research and Application Center (ATADEM) laboratory of Atatürk University. The water intake and pellet intake of the animals were ad libitum. Before starting the experiment, the rats were kept in cages for 1 week at normal room temperature (22 °C) in order to adapt to the environment. The ethics committee approval of the study was approved by the Animal Experiments Local Ethics Committee of Atatürk University with the document dated 11 January 2022 and numbered E-42190979-000-2200008858. A total of 72 male rats were randomized into 6 groups (5 experimental groups and 1 control group, 12 animals in each group). Before starting the experiment, all of the animals were weighed, and their weights were recorded. Imidacloprid (5.7 mg/kg), acetamiprid (12.4 mg/kg) and S. Boulardii (1 × 109 CFU/day) were given orally to the animals, divided into groups, for 90 days [5 (link),27 (link),28 (link)].
The animals were sacrificed 90 days after injection. The rats were decapitated rapidly under deep anesthesia (Sevoflurane). One testis of each animal in each group was collected and fixed in 10% neutral formaldehyde for immunohistochemical analysis (Sigma-Aldrich, St. Louis, MO, USA).
The animals were sacrificed 90 days after injection. The rats were decapitated rapidly under deep anesthesia (Sevoflurane). One testis of each animal in each group was collected and fixed in 10% neutral formaldehyde for immunohistochemical analysis (Sigma-Aldrich, St. Louis, MO, USA).
3
EGF-Induced Cell Invasion Assay
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EGF (20 ng/mL) treated LoVo cells were seeded at a density of 1 x 105 on matrigel coated chamber (BD bioscience) with or without treatment with KY1022 (20 μM). Cells were allowed to invade for 18 hours. After clearing the cells on the inner surface of the chamber, the cells on the outer surface were fixed using 10% neutral formaldehyde (Sigma) for 15 minutes and stained with crystal violet for 15 minutes. The chambers were dipped in distilled water to remove the excess staining and allowed to dry. Representative images were captured by microscope (TE-2000U, Nikon). Mean ± s.d. are reported, based on 3 biological replicates. Mean ± s.d. are reported, based on 3 or 5 biological replicates.
4
Cytotoxicity Evaluation of KY1022 in CRC Cells
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CRC cell lines (SW480, HCT15 and LoVo) were plated at a density of 7 × 103 cells in 96 well plate, and HEK293 cells were seeded at a density of 1 × 104 cells per 96 well plate. The cells were then treated with dimethyl sulfoxide (DMSO) or KY1022 for 24, 48 and 72 hours. 3-(4 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; AMRESCO) was added to each well at a concentration of 0.25 mg/mL. After incubation for 2 hours at 37°C, insoluble purple formazan was obtained by removing the media and incubated in 100 μL DMSO for 1 hour. The absorbance of the formazan product was measured by FLUOstar OPTIMA (500 nm). For the colony formation assay, LoVo cells were seeded at a density of 500 cells per well in 12-well plates. The cells were treated with DMSO or KY1022 for 14 days. Media containing 5% serum was changed every 3 days. At the end of the experiment, cells were fixed in 10% neutral formaldehyde (Sigma) for 30 minutes in room temperature and stained with 0.5% crystal violet in 20% ethanol for 30 minutes. Mean ± s.d. are reported, based on 3 biological replicates.
5
Oil Red O Staining of Lipids
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The cells were washed with warm phosphate-buffered saline (Sigma-Aldrich), fixed with neutral formaldehyde (10%, Sigma-Aldrich) at room temperature for 2–3 h, then rinsed quickly with isopropanol (60%, Sigma-Aldrich) and allowed to dry. After that, we used Oil Red O (Sigma-Aldrich; 0.5% w/v diluted 3:2 with double-distilled water) to stain the cells and incubated the cells at room temperature for 2–3 h prior to 3–4 washes with distilled water. Images were then acquired by means of the Olympus microscope IX71 (Olympus, Center Valley, PA, USA). To release Oil Red O from steatosis staining, 500 μL of isopropanol was added into each well prior to incubation for 10 min at room temperature. After being transferred to a 96-well plate, the optical density of the isopropanol solution at a wavelength of 490 nm was determined by means of a microplate reader (Versa Max).
6
Imazamox Herbicide Toxicology in Rats
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A total of 50 male Sprague-Dawley rats (mean weight 300 ± 10 g SD) were used in this study. Animals were randomly assigned into 10 groups (n = 5/group) including control, three low dose groups (12 mg/kg bw; 24 h, 48 h, 72 h), three middle dose groups (24 mg/kg bw; 24 h, 48 h, 72 h) and three high dose groups (36 mg/kg bw; 24 h, 48 h, 72 h). All doses were calculated based on the LD50 and from the current/recent bibliography. Furthermore, we took into account the NOAEL doses from reports of risk assessment [9 ]. After a 5-day adaptation period, the imazamox-based herbicide was mixed with 0.9% isotonic sodium chloride to allow administration of a 12, 24 and 36 mg/kg bw imazamox equivalent dose. A volume of 1 mL was injected intraperitoneally. The animals were sacrificed at 24, 48 or 72 h following injection. Following imazmox administration, blood samples were collected by cardiac puncture into vacuum tubes with no anticoagulant (Vacutainer, BD-Plymouth, UK). Blood samples were centrifuged at 3000g for 10 min at room temperature to isolate serum and stored at −20 °C until analysis. Rats were decapitated rapidly under deep anesthesia (Sevoflurane, USA), livers and pancreases were excised and fixed in 10% neutral formaldehyde (Sigma, USA).
7
Counting Sensorimotor Cortical Neurons
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Three weeks after surgery, rats were anesthetized with ketamine (0.05 mL/kg) and xylazine (0.01 mg/kg) and then transcardially infused with 0.9% saline followed by 10% ice-old neutral formaldehyde (Sigma, St. Louis, MO). Their brains were removed, placed in 10% neutral formaldehyde overnight at room temperature, and processed for paraffin histology. Then, 5-μm sections were stained with neuron-specific enolase (NSE) using the method described earlier. The cortical neurons in the sensorimotor area of the forelimb were counted in five randomly selected frontal sections by investigators who were blinded to the allocation of treatment groups. These neurons were identified by their location, size, and NSE staining. The neuron density of each group (three animals) was expressed as the mean number of neurons per 10,000 μm2.
8
Histological Analysis of Rat Intestinal Mucus
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The Wistar rat intestine samples were processed as described by Naour et al. [24 (link)]. Briefly, the experimental animals were euthanized, and intestinal tissue was dissected and placed in ice-cold phosphate buffered saline (PBS). Intestine samples were rapidly rinsed in PBS and flushed using ice-cold modified Bouin fixative (5% acetic acid, 50% ethanol in deionized water). The Swiss-Roll technique was then applied, and samples were placed in tissue processing cassettes and fixed overnight in 10% (v/v) neutral formaldehyde (Sigma-Aldrich, St. Louis, MO, USA).
Tissue samples were then processed in an automatic tissue processor, where paraffin was embedded and sectioned. Tissue sections were then stained with hematoxylin-Eosin stain to characterize tissue architecture and Alcian blue staining to visualize mucins. Tissue sections were then evaluated by a pathologist blinded by the experimental design.
The thickness of the mucus layer was measured using ImageJ software (NIH, Bethesda, MD, USA). Briefly, the histological images were processed, and the microscopy size bar length was used for the normalization of the pixel count. Then, subsequently, the Alcian blue-stained mucus was measured in at least five different images, and the average length (mm) was then determined.
Tissue samples were then processed in an automatic tissue processor, where paraffin was embedded and sectioned. Tissue sections were then stained with hematoxylin-Eosin stain to characterize tissue architecture and Alcian blue staining to visualize mucins. Tissue sections were then evaluated by a pathologist blinded by the experimental design.
The thickness of the mucus layer was measured using ImageJ software (NIH, Bethesda, MD, USA). Briefly, the histological images were processed, and the microscopy size bar length was used for the normalization of the pixel count. Then, subsequently, the Alcian blue-stained mucus was measured in at least five different images, and the average length (mm) was then determined.
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