Osteogenesis was induced in BMSCs with treatment of imperatorin at different concentrations. After 7 days, cells were fixed with 4% paraformaldehyde solution and permeabilized with 0.2% Triton X‐100 for 15 minutes and non‐specific binding was blocked with 10% goat serum solution. Cells were then incubated overnight at 4°C with the primary antibodies: COL1A1, RUNX2, OCN, BMP2 and β‐catenin, after which they were washed with PBS and incubated for 1 hour at room temperature with an appropriate Alexa fluorescent‐conjugated antibody (Molecular Probes, Life Technologies) in 1:400 dilution. Finally, the plate was stained with DAPI Fluoromount‐G and anti‐fluorescence quenched with DAPI Fluoromount‐G™ (YEASEN). Stained sections were observed under fluorescence microscope (Olympus BX53; Olympus Corporation).
Dapi fluoromount g
Manufactured by Yeasen
Sourced in China
DAPI Fluoromount-G is a mounting medium for fluorescence microscopy. It is designed to preserve fluorescence signals and provide long-term stability for fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Rhodamine coupled phalloidin, by Yeasen (2 mentions)
A1 plus, by Nikon (1 mentions)
Fixation permeabilization solution, by BD (1 mentions)
Tcs sp8, by Leica (1 mentions)
Af3667, by R&D Systems (1 mentions)
Confocal microscope, by Leica (1 mentions)
Bovine serum albumin (bsa), by Yeasen (1 mentions)
Nile red, by Yuanye Bio-Technology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Coumarin 6, by Yuanye Bio-Technology (1 mentions)
Hoechst 33342 staining kit, by Beyotime (1 mentions)
Porphyromonas gingivalis lps, by InvivoGen (1 mentions)
Dioscin, by Chengdu Must Bio-Technology (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Mitochondrial isolation kit, by Beyotime (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
12 protocols using dapi fluoromount g
1
Osteogenic Differentiation of BMSCs
2
Immunofluorescence Imaging of RNA Polymerase II
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At the end of the culture period, the cells were fixed with 4% paraformaldehyde (PFA) for 20 min. Next, the cells were washed in PBS three times for 2 min each. The cells were incubated with 5% BSA in PBS containing 0.1% Triton X-100 at 37°C for 30 min and then incubated overnight at 4°C with a primary antibody against RNA polymerase II (1:100, Abcam). The cells were washed three times with PBS and incubated with Alexa Fluor 488 donkey anti-rabbit secondary antibodies (1:1000) for 1 hour at 37°C. The cells were rinsed three times with PBS and incubated with DAPI Fluoromount-G for 10 min (Yeasen, Shanghai). For staining of F-actin, rhodamine-coupled phalloidin was used (Yeasen, Shanghai). All images were captured on a confocal fluorescence microscope (Nikon, A1 PLUS, Tokyo, Japan).
3
Microscopic Quantification of Neutrophil Extracellular Traps
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophil extracellular traps (NETs) were counted in mouse blood samples by conventional Giemsa microscopy or immunofluorescence.
In Giemsa-stained thin smears, NETs were identified under a 100× oil objective lens as extracellular structures with staining of decondensed chromatin associated with fragmented neutrophil-like cells or small granules12 (link).
For immunofluorescence, blood cells were collected, washed once in FACS buffer, fixed and permeabilized with BD Fixation/Permeabilization solution for 10 min at 4 °C. Then, the cells were washed with BD Perm/ Wash™ buffer and incubated with MPO antibody (1:200, R&D, AF3667) and citrullinated histone H3 antibody (1:200, Abcam, ab5103) overnight at 4 °C. Cells were washed once in FACS buffer. The secondary antibodies used were Alexa Fluor 488 conjugated anti-goat IgG (1:1000, Abcam, ab150129) and Alexa Fluor 555 conjugated anti-rabbit IgG (1:1000, CST, 4413). After staining for 1 h at RT, samples were washed once in FACS buffer and stained with DAPI Fluoromount-G™ (Yeasen, 36308ES20). Images were obtained by laser confocal microscope (Leica TCS SP8) using a 63× oil objective lens. For quantification, at least five random fields per sample were analyzed. The average value under each field was taken as the sample value.
In Giemsa-stained thin smears, NETs were identified under a 100× oil objective lens as extracellular structures with staining of decondensed chromatin associated with fragmented neutrophil-like cells or small granules12 (link).
For immunofluorescence, blood cells were collected, washed once in FACS buffer, fixed and permeabilized with BD Fixation/Permeabilization solution for 10 min at 4 °C. Then, the cells were washed with BD Perm/ Wash™ buffer and incubated with MPO antibody (1:200, R&D, AF3667) and citrullinated histone H3 antibody (1:200, Abcam, ab5103) overnight at 4 °C. Cells were washed once in FACS buffer. The secondary antibodies used were Alexa Fluor 488 conjugated anti-goat IgG (1:1000, Abcam, ab150129) and Alexa Fluor 555 conjugated anti-rabbit IgG (1:1000, CST, 4413). After staining for 1 h at RT, samples were washed once in FACS buffer and stained with DAPI Fluoromount-G™ (Yeasen, 36308ES20). Images were obtained by laser confocal microscope (Leica TCS SP8) using a 63× oil objective lens. For quantification, at least five random fields per sample were analyzed. The average value under each field was taken as the sample value.
4
Immunofluorescence Staining of METTL16
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GC cells transfected with point mutation plasmids were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde at room temperature for 15 min, permeabilized with 0.5% Triton X-100 for 20 min and blocked with 3% BSA (PBS) for 30 min. Then the samples were incubated at 4 °C overnight with the primary antibody METTL16 (Abclonal Technology, Wuhan, China) diluted in 3% BSA (PBS) overnight. Subsequently, the cells were washed three times with PBS and incubated with fluorescein-labeled secondary antibodies diluted in PBS for 1 h at room temperature. The cells were stained and sealed with DAPI Fluoromount-G (Yeasen, Shanghai, China), and observed using a confocal microscope (Leica, Wetzlar, Germany).
5
Fluorescent Labeling Agents for Cell Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BSA and DAPI Fluoromount-G were purchased from Yeasen Biotechnology Co., Ltd. (Shanghai, China). In addition, 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) and ethanol were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Nile red and coumarin-6 were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Roswell Park Memorial Institute 1640 medium (RPMI 1640), Dulbecco’s modified Eagle’s medium (DMEM), trypsin, fetal bovine serum (FBS), and 1% penicillin–streptomycin were purchased from Gibco. ABP was purchased from Jiangsu Hengrui Pharmaceuticals Co., Ltd. (Lianyungang, China).
6
Mitochondrial Dysfunction and Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dioscin was sourced from Chengdu Must Bio-technology Co., Ltd. Porphyromonas gingivalis-LPS was procured from InvivoGen. The ROS Assay Kit, Mito-Tracker Red CMXRos, Hoechst 33342 staining kit, and Mitochondrial Isolation kit were obtained from Beyotime. MitoSOX™ Red was from Thermo Fisher. DAPI Fluoromount-G™ was from Yeasen. Mitoquinone (MitoQ) was bought from MCE.
7
Immunofluorescence Assay for Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed on the cover clip with 4% paraformaldehyde for 15 min, and permeabilized with 0.2% Triton X-100 in PBS for 5 min. Fixed cells were then blocked with 3% BSA in PBS for 1 h, before incubation with anti-DHX15 or anti-MYC antibody at 4°C overnight. Cells were subsequently stained with Fluo-555 (1:200, Cell Signaling Technology) conjugated anti-rabbit secondary antibody or Fluo-488 (1:200, Cell Signaling Technology) conjugated anti-mouse secondary antibody for 1 h. After washing three times with PBS in dark, cells were mounted with DAPI Fluoromount-G (Yeasen). Immunofluorescence signals were detected by confocal microscopy (Nikon Eclipse, Japan).
8
Immunofluorescence Staining of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain tissues were fixed with 4% paraformaldehyde (PFA) for 10 min. For IF staining, the tissue slices were permeabilized with 0.1% Triton X-100 for 10 min, blocked with 1% BSA for 1 h at 37 °C, and then incubated with antibodies against AQP4 (Servicebio, Wuhan, China, GB12529, 1:200), CD31 (R&D, AF3628, 1:200), and Aβ (Abcam, ab201060, 1:200) at 4 °C overnight. The next day, tissue slices were incubated with fluorescent secondary antibodies for 1 h at room temperature. The nuclei were stained with DAPI Fluoromount-G™ (Yeasen, Shanghai, China, 36308ES20) for 10 min prior to imaging.
After the same pre-treatment as before, the cell slides were incubated with antibodies against CD163 (ProteinTech Group, Chicago, IL, USA, Cat No: 16646-1-AP, 1:400) and iba-1 (WAKO, 011-27991) and the next steps are also consistent with the above. Representative regions and cells were selected and photographed.
After the same pre-treatment as before, the cell slides were incubated with antibodies against CD163 (ProteinTech Group, Chicago, IL, USA, Cat No: 16646-1-AP, 1:400) and iba-1 (WAKO, 011-27991) and the next steps are also consistent with the above. Representative regions and cells were selected and photographed.
9
Immunofluorescence Staining for Brain Slices
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining was carried out as described previously (25 (link)). In brief, 25-μm-thick frozen coronal brain slices were cut, blocked (5% donkey serum), and incubated in primary antibodies (diluted in 0.3% Triton X-100 of PBS containing 1% goat or donkey serum) at 4°C overnight. After washing, slices were incubated in secondary antibodies at room temperature for 2 hours. Brain slices were finally mounted with DAPI Fluoromount-G (36308ES20, Yeasen, Shanghai, China) after washing. ImageJ and Imaris software were used to analyze images that were captured by a Nikon A1 confocal microscope. Source code for immunofluorescence quantitative analysis (Fig. 6 and fig. S8) is available at https://zenodo.org/record/8238087 .
10
Quantifying Neuronal Apoptosis via TUNEL Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TUNEL assay was performed to measure neuronal apoptosis using a kit purchased from Beyotime Institute of Biotechnology, according to the manufacturer's instructions. After dewaxing in xylene for 5 min twice, anhydrous ethanol for 5 min, 90% ethanol for 2 min, 70% ethanol for 2 min and distilled H2O for 2 min (all at room temperature), paraffin-embedded sections were treated for 20 min with DNase-free proteinase K (20 µg/ml) at 37˚C. Subsequently, the sections were incubated for 1 h TUNEL working solution at 37˚C in the dark. DAPI Fluoromount-G™ (Shanghai Yeasen Biotechnology Co., Ltd.) was utilized for counterstaining for 10 min at room temperature before observation under a fluorescence microscope (Olympus Corporation). The apoptotic index was determined as (TUNEL-positive cells)/(total cells) x100.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!