The effect of Mel-MBG/SA extract on the production of ECM in NPCs was evaluated through cellular immunofluorescence using a specific marker for nucleus pulposus (COL2A1). Inflammatory microenvironment was established by supplementing the medium with 5 ng/mL of IL-1β for a specified duration. NPCs were seeded in 6-well plates and treated with normal growth medium or Mel-MBG/SA extract for 3 days. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X, and blocked with 5% bovine serum albumin (BSA). The cells were then incubated overnight at 4 °C with a primary antibody against COL2A1 (1:250, Abcam, USA), followed by incubation with Alexa Fluor 594-conjugated secondary antibody (Abcam) for 1 h. DAPI staining (Solarbio, China) was performed for 5 min, and the cells were then visualized using a fluorescent microscope (Olympus IX83, Japan).
Dapi staining
Manufactured by Solarbio
Sourced in China
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that selectively binds to DNA, allowing for the visualization of cell nuclei. It is commonly used in fluorescence microscopy applications.
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Lab products found in correlation
Alexa fluor 594 conjugated secondary antibody, by Abcam (1 mentions)
Sodium citrate antigen retrieval solution, by Solarbio (1 mentions)
Goat anti rabbit igg fitc, by Solarbio (1 mentions)
One step tunel apoptosis assay kit, by Roche (1 mentions)
A1r a1, by Nikon (1 mentions)
Cy3 goat anti rabbit igg, by ABclonal (1 mentions)
Fitc goat anti mouse igg, by ABclonal (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated igg, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Nikon (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Goat serum, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Leagene (1 mentions)
6 protocols using dapi staining
1
Evaluating ECM Production in NPCs
2
Immunofluorescent Detection of VASA in Testicular Tissue
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Testicular tissue sections were dewaxed and rehydrated, followed by Sodium Citrate Antigen Retrieval Solution (Solarbio, Beijing, China) at 95 °C for 15 min. Tissue permeabilization was conducted using phosphate buffered saline (PBS) containing Triton X-100. Tissue sections were blocked with 10% goat serum diluted in PBS at room temperature for 30 min. The VASA antibody (Gene Tex, Irvine, CA, USA) was diluted 1:500 and incubated at room temperature for 30 min, followed by incubation at 4 °C for 24 h, then returned to room temperature for an additional 30 min. Sections were washed five times with PBS for 3 min each. Goat Anti-Rabbit IgG/FITC (Solarbio, Beijing, China) was diluted 1:200 and incubated at room temperature for 1 h. This was followed by five PBS washes for 3 min each. DAPI staining (Solarbio, Beijing, China) solution was diluted 1:1000 and applied for 1 min, then sections were washed 3–5 times with PBS. Slides were mounted with 50% glycerol and observed and photographed under a fluorescence microscope.
3
Cardiac Apoptosis Detection in Mice
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At the end of the treatment period, all mice were euthanized with an overdose of sodium pentobarbital (200 mg/kg, i.p.). Animal death was verified by observation of cardiac arrest and pupil enlargement for 1 min. The humane endpoints established in this study were as follows: Impaired ambulation that prevented animals from reaching food or water; excessive weight loss and extreme emaciation; lack of physical or mental alertness; difficult labored breathing; and prolonged inability to remain upright. Animals were observed a minimum of twice daily, with more frequent observations immediately after dosing and when increased morbidity or mortality was expected (17 (link)). The heart was then removed and fixed in 10% formalin for 24–48 h at 4°C. Formalin-fixed heart tissues were then embedded in paraffin and sections were cut (~4 µm thick). Cardiomyocyte apoptosis was detected using a one-step TUNEL Apoptosis assay kit at 37°C for 1 h (Roche Diagnostics GmbH), according to the manufacturer's protocol, followed by DAPI staining (10 µg/ml; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 2 min. Anti-fade mounting medium was then added (cat. no. P0126; Beyotime Institute of Biotechnology) to each slide. Images were obtained from five fields per slide using confocal microscopy (magnification ×400; NIKON A1R/A1; Nikon Corporation).
4
Immunofluorescence Staining of Rat Cerebral Tissue
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Immunofluorescence staining was performed according to our prior procedure [28 (link)]. Briefly, the cerebral tissues of rats were embedded in OCT and sliced by a cryosectioning machine (Leica, Germany) into coronal sections of 20 µm thickness. Cultured astrocytes were immobilized with paraformaldehyde (4%). Entire samples were blocked with goat serum albumin (10%) and overnight incubation of the samples at 4 °C was carried out using the primary antibodies shown in Table 2 . Subsequently, the samples were incubated with FITC Goat Anti-Mouse IgG or Cy3 Goat Anti-Rabbit IgG (1:200, ABclonal, China) secondary antibodies, followed by the DAPI staining (Solarbio, China). A fluorescence microscope (Leica, Germany) was utilized for the imaging, and Image J was employed for the image analysis.
5
Immunofluorescence Analysis of HepG2-IR Cells
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The HepG2-IR cells treated with BBR were fixed with 4% formaldehyde and permeabilized in 0.5% Triton X-100. The fixed HepG2-IR cells were washed with PBS and blocked with 5% bovine serum albumin in PBS for 60 min at room temperature. Fixed HepG2-IR cells were then incubated with the primary antibodies overnight at 4°C, then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody (1 : 1000; Life Technologies, Waltham, MA, USA) or Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (1 : 200; ab1501115, Abcam, UK) for 1 h at room temperature, followed by DAPI staining (Solarbio Life Sciences, China) in the dark. Images were taken by a fluorescence microscope.
6
Immunofluorescent Quantification of Microglia Morphology
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The brain slices were fixed in 4% paraformaldehyde (Leagene) for 24 h and then dehydrated in serial 15% and 30% sucrose solutions. The brain tissue was then sliced into 20-μm-thick coronal sections. After rinsing the brain sections with PBS for residual embedding medium (OCT), the sections were blocked with 5% goat serum (Beyotime) and 1% Triton-X 100 (Beyotime) for 1.5 h, followed by incubation with primary antibodies against NeuN (Rabbit, 1:200, Abcam) and Iba-1 (Rat, 1:500, Synaptic Systems, 234017) overnight at 4°C, followed by treatment with Alexa Fluor 594-conjugated IgG (1:200, Invitrogen) and Alexa Fluor 488-conjugated IgG (1:200, Invitrogen) secondary antibodies for 1 h at room temperature. Next, PBS was used to rinse the sections thrice, followed by DAPI staining (Solarbio, Beijing, China) for 10 min. Subsequently, a confocal microscope (Nikon) was employed for observation. All fluorescent image data was quantified by using Image J. For microglia morphology data collection, the fluorescence photomicrographs were converted into representative binary and skeletonized images and then analyzed using the ImageJ plugins AnalyzeSkeleton (2D/3D) and FracLac.
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