Alpha-amylase from human salivary, alpha-glucosidase (1 unit/mL, Sigma), the ACE solution from rabbit lung, 2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid sulfonic acid) (ABTS·+), dibutylphthalate polystyrene xylene (DPX), 4-dimethylamino cinnamaldehyde (DMACA), 3,5-dinitrosalicylic acid (DNS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric chloride, hippuroyl-His-Leu hydrate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), Melanin from Sepiaofficinalis, p-nitrophenyl alpha-d -glucopyranoside (pNPG), phosphate buffer, potassium ferricyanide, potassium permanganate, potassium persulfate, potassium phosphate buffer, sodium borate buffer, sodium carbonate, sodium hydroxide, sodium potassium tartrate, starch, and xylene were from Sigma Chemical Company (St. Louis, Missouri, USA). Analytical-grade acetic acid, acetone, chloroform, ethanol, ethyl acetate, hydrochloric acid, hydrogen peroxide, and methanol were from Honeywell (Seelze, Hanover, Germany).
Dibutylphthalate polystyrene xylene (dpx)
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Macao, Ireland, Spain, Italy, Australia, Poland, India, Sao Tome and Principe, Canada
DPX is a laboratory instrument designed for the analysis and separation of chemical compounds. It functions as a high-performance liquid chromatography (HPLC) system, capable of accurately and efficiently separating and identifying the individual components within a sample. The DPX system provides essential analytical capabilities for researchers and scientists in various fields, including pharmaceutical development, environmental analysis, and quality control.
Automatically generated - may contain errors
Lab products found in correlation
Cresyl violet, by Merck Group (15 mentions)
Xylene, by Merck Group (12 mentions)
Hematoxylin, by Merck Group (12 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (10 mentions)
Vectastain abc kit, by Vector Laboratories (9 mentions)
3 3 diaminobenzidine (dab), by Merck Group (9 mentions)
Fluorescence microscope, by Olympus (6 mentions)
Vectastain elite abc kit, by Vector Laboratories (6 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Bx51 microscope, by Olympus (6 mentions)
Lsm 880 confocal microscope, by Zeiss (6 mentions)
Zen blue software, by Zeiss (6 mentions)
Epifluorescent microscope, by Zeiss (5 mentions)
Abc elite kit, by Vector Laboratories (5 mentions)
Cryostat, by Leica (5 mentions)
Phosphate buffered saline (pbs), by Merck Group (5 mentions)
Paraformaldehyde (pfa), by Merck Group (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Haematoxylin, by Merck Group (5 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (5 mentions)
448 protocols using dibutylphthalate polystyrene xylene (dpx)
1
Comprehensive Biochemical Assay Protocol
2
Fluoro-Jade B Staining Protocol for Quantifying Neuronal Degeneration
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F-J B is used as a marker of cellular degeneration. F-J B histofluorescence was carried out in order to examine neuronal damage/death following TFI. As described previously [12 (link),26 (link)], in brief, the sections were incubated in 0.06% potassium permanganate (KMnO4) (Sigma-Aldrich Co.) for 20 min on an orbital shaker. After washing for two minutes, these sections were reacted in 0.0004% F-J B (Histochem, Jefferson, AR, USA) for 30 min. Thereafter, the brain sections were washed three times for two minutes and placed on a slide warmer until they were fully dried. The slides were cleared using xylene for one minute and coverslipped with dibutylphthalate polystyrene xylene (Sigma-Aldrich Co.).
The count of F-J B positive cells (neurons) was performed to evaluate TFI-induced neuronal death in the CA1 region. In short, according to published methods [4 (link),24 (link)], the image of F-J B positive cells was taken at the magnification of 20X using epifluorescent microscope (Carl Zeiss, Oberkochen, Germany), equipped with a digital camera (DP72) (Olympus), at 450–490 nm of wavelength. F-J B positive cells were taken using image capture software (cellSens Standard) (Olympus). The total number of F-J B positive cells was counted in the same way described above (count of NeuN immunoreactive neurons).
The count of F-J B positive cells (neurons) was performed to evaluate TFI-induced neuronal death in the CA1 region. In short, according to published methods [4 (link),24 (link)], the image of F-J B positive cells was taken at the magnification of 20X using epifluorescent microscope (Carl Zeiss, Oberkochen, Germany), equipped with a digital camera (DP72) (Olympus), at 450–490 nm of wavelength. F-J B positive cells were taken using image capture software (cellSens Standard) (Olympus). The total number of F-J B positive cells was counted in the same way described above (count of NeuN immunoreactive neurons).
3
Assessing Neuronal Degeneration in Hippocampus
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FJB histofluorescence was performed to examine neuronal death (loss) in the hippocampus with reference to some previous reports [26 (link),27 (link)]. The brains sections were put onto the slide glasses, which were coated with gelatin. These sections were soaked in 0.06% KMnO4 (Sigma-Aldrich Co., St. Louis, MO, USA) for 20 min and briefly washed. After washing, these sections were soaked in 0.0004% FJB (Histo-chem Inc., Jefferson, AR, USA) for 30 min and rinsed. Thereafter, these sections were placed onto slide warmer until they were completely dried. The reacted sections were finally cleared in xylene and coverslipped with dibutyl phthalate polystyrene xylene (Sigma-Aldrich Co., St. Louis, MO, USA).
In order to count numbers of FJB positive cells, five sections per gerbil were chosen. According to a paper [28 (link)] with some modification, the stained sections were observed using epifluorescent microscope (Carl Zeiss, Göttingen, Germany) with blue excitation fluorescent filter (wavelength of 450–490 nm). Images of FJ B positive cells, which underwent degeneration (bright fluoresce) when compared with the background [26 (link)], were captured and counted in 250 × 250 μm at the middle of the CA1 region. Finally, the mean number of FJB positive cells was calculated using NIH Image 1.59 software (NIH, Bethesda, Rockville, MD, USA).
In order to count numbers of FJB positive cells, five sections per gerbil were chosen. According to a paper [28 (link)] with some modification, the stained sections were observed using epifluorescent microscope (Carl Zeiss, Göttingen, Germany) with blue excitation fluorescent filter (wavelength of 450–490 nm). Images of FJ B positive cells, which underwent degeneration (bright fluoresce) when compared with the background [26 (link)], were captured and counted in 250 × 250 μm at the middle of the CA1 region. Finally, the mean number of FJB positive cells was calculated using NIH Image 1.59 software (NIH, Bethesda, Rockville, MD, USA).
4
Immunohistochemical Analysis of Aortic Tissue
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Blocks of paraffin-embedded aorta tissue were sectioned to a thickness of 7 µm, placed on a coating slide and dried at 40 °C for 24 h. Slides were then deparaffinized and incubated in 0.3% H2O2 (Sigma-aldrich, Missouri, MO, USA) for 30 min. Slides were subsequently rinsed three times with PBS and incubated in normal animal serum to block non-specific binding and incubated with primary antibodies (Table S2 ) at 4 °C, followed by three additional rinses with PBS. Slides were then treated with biotinylated secondary antibodies from the ABC kit (dilution rate 1:200; Vector Laboratories, Burlingame, CA, USA), incubated for 1 h with blocking solution, and rinsed three times with PBS. Slides were left to react with DAB substrate for 15 min, followed by mounting with a cover slip and dibutylphthalate polystyrene xylene (Sigma-aldrich, Missourti, MO, USA) mounting solution. Images were detected using a light microscope (Olympus, Tokyo, Japan) and quantification of the intensity of the brown color was performed using Image J software (NIH, Maryland, MD, USA) [45 (link),46 (link)].
5
Fluorescent Staining of Brain Tissue
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The brain tissue slides were hydrated in distilled water for 1 minute and immersed in 0.06% potassium permanganate for 15 minutes at room temperature. The slides were stained with 0.001% FJB (Histo-Chem Inc., Jefferson, AR, USA) solution for 30 minutes. After washing the stained slides three times in distilled water for 5 minutes, the slides were dried at 55℃ in the dark for at least 35 minutes and mounted with dibutyl phthalate polystyrene xylene (Sigma-Aldrich Co., Saint Louis, MO, USA). FJB-stained brain tissue images were obtained with light microscopy (Carl Zeiss, Oberkochen, Germany) at ×20 magnification. The stained brain tissues were observed at 450–490 nm using a fluorescence microscope.
6
Fluoro-Jade C Staining of Brainstem Sections
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Brainstem sections were randomly selected and stained with Fluoro-Jade C (Chemicon, Tokyo, Japan) following the manufacturer’s protocol. In brief, the slices were immersed in a solution of 80% EtOH with 1% of NaOH for 5 min, then rinsed for 2 min in 70% EtOH and 2 min in distilled water, and incubated for 10 min with 0.06% permanganate solution. After washing with water, the slices were transferred to a solution of 0.0001% of Fluoro-Jade C dissolved in 0.1% acetic acid and were incubated for 10 min. Then, the slices were washed with water, counterstained with DAPI, and mounted with dibutylphthalate polystyrene xylene (Sigma). Confocal images were taken within several hours of the staining to avoid fluorescence loss.
7
Immunohistochemical Analysis of Distal Colon
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The distal portion of the colons from mice maintained on custom diets as well as those treated with CAY10397 or 15d-PGJ2 and their respective vehicle controls were stored in 10% (v/v) buffered formalin. The sections were prepared at the Histopathology Core Facility, Animal Diagnostic Laboratory at Penn State University, University Park, PA. To detect Zo-1, sections were deparaffinized, followed by antigen-retrieval, and blocking with 5% goat serum. The sections were incubated in rabbit anti-Zo-1 antibody (1:500; Abcam) overnight at 4°C. The sections were washed in TBS and 0.5% hydrogen peroxide used to block any endogenous peroxidase. The sections were incubated in goat anti-rabbit secondary antibody (Vector Laboratories) for 1 h followed by incubation in ABC reagent (Vector Laboratories) for 45 min at room temperature. DAB substrate kit (Vector Laboratories) was used to develop peroxidase activity and the sections were dehydrated and mounted in dibutylphthalate polystyrene xylene (Sigma). The slides were imaged using an OLYMPUS DP74 microscope and OLYMPUS CellSens Standard software at magnification 10x. The length of well-oriented crypts of the distal colon of individual mice was also measured using the OLYMPUS CellSens Standard software.
8
Identifying Inferior Colliculus Subdivisions
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A detailed description of the methods used for identification of IC subdivisions has been previously reported (Nakamoto et al., 2013a (link)). Briefly, one series of sections from each animal was stained for NADPH-d activity (Dawson et al., 1991 (link)). The stained sections were mounted on slides, dried overnight, and then coverslipped with DPX (Aldrich Chemical Company, Inc., Milwaukee, WI, USA). The NADPH-d stain reflects the distribution of neuronal nitric oxide synthase and can be used to distinguish IC subdivisions in guinea pigs (Coote and Rees, 2008 (link)).
9
Confocal Microscopy for Immunofluorescence Labeling
10
Confocal Microscopy for Immunofluorescence Analysis
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For confocal microscopy, detection of immunofluorescence-labeled antigens utilized two combinations of secondary antibodies. The first combination included secondary antibodies that were fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (1:200; Jackson Immunoresearch) for MGL or FAAH detection; tetramethylrhodamine-5-isothiocyanate (TRITC)-conjugated donkey anti-guinea pig IgG (1:200; Jackson Immunoresearch) for CB1 detection and Cy5-conjugated donkey anti-mouse IgG (1:200; Jackson Immunoresearch) for DβH or NET detection. Tissue sections underwent serial dehydration, were mounted on slides, and coverslipped using DPX (Aldrich). Slides were then viewed using an Olympus IX81 inverted confocal microscope (Hatagaya, Shibuya-Ku, Tokyo, Japan), with helium and argon laser excitation wavelengths of 488, 543 and 635. The microscope is also equipped with filters (DM 405–44, BA505-605, and BA 560–660) and the Olympus Fluoview ASW FV1000 program. Fluorescence confocal images were assembled and adjusted for brightness and contrast in Adobe Photoshop.
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