MES23.5 cells (2x104 cells/well) were plated in 24-well plates, fixed with 4% paraformaldehyde (Macklin, Inc.) for 1 h at room temperature, and blocked in 1% BSA (Beijing Solarbio Science & Technology Co., Ltd.) for 0.5 h at room temperature. Next, the cells were incubated in the presence of a primary antibody against TH (cat. no. ab137869; 1:100; Abcam) at 4˚C overnight, followed by incubation with the Alexa Fluor® 488-labelled goat anti-rabbit secondary antibody (cat. no. ab150077; 1:200; Abcam) for 1 h at room temperature. The nuclei were counterstained with DAPI for 5 min at room temperature. The results were observed under a fluorescence microscope (magnification, x200; Olympus Corporation).
Paraformaldehyde (pfa)
Manufactured by Maclin Biochemical Technology
Sourced in China
Paraformaldehyde is a solid polymer of formaldehyde. It is a white, crystalline solid that is commonly used as a fixative and preservative in biological and chemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (2 mentions)
Phenol, by Maclin Biochemical Technology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Solarbio (2 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Ab137869, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
Fitc goat anti rabbit igg, by EarthOx (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Enhanced immunostaining permeabilization buffer, by Beyotime (1 mentions)
Gel documentation system, by Thermo Fisher Scientific (1 mentions)
Alcian blue 8 gx, by Solarbio (1 mentions)
Urea, by Maclin Biochemical Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab150113, by Abcam (1 mentions)
Sc 393325, by Santa Cruz Biotechnology (1 mentions)
Fluorescein isothiocyanate conjugated goat anti rabbit igg antibody, by Boster Bio (1 mentions)
P toluenesulfonic acid monohydrate, by Maclin Biochemical Technology (1 mentions)
27 protocols using paraformaldehyde (pfa)
1
Immunofluorescence Assay for TH Expression
2
Immunofluorescence Assay for YAP1
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Both TCam-2 and NTERA-2 cells were fixed with 4% paraformaldehyde (Macklin, China) for 5 min and permeabilized with 0.1% Triton X-100 (Sigma–Aldrich) for 5 min at room temperature. After that, the cells were blocked with 5% BSA in PBS for 1 h and incubated with primary antibodies against YAP1 (1:100, ABclonal). Cells were then labelled with FITC goat anti-rabbit IgG (1:200, Earthox). Nuclei were stained with DAPI (1:400, Solarbio). Fluorescence images were acquired by using an inverted fluorescence microscope (Olympus).
3
Colorimetric TUNEL Assay for PC12 Cell Apoptosis
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A colorimetric TUNEL Apoptosis Assay kit (cat. no. C1088; Beyotime Institute of Biotechnology) was utilized to observe PC12 cell apoptosis. The cells (5x105/well) seeded into a 24-well plate were fixed with 4% paraformaldehyde (Shanghai Macklin Biochemical Co., Ltd.) at room temperature for 30 min, and incubated with Enhanced Immunostaining Permeabilization Buffer (Beyotime Institute of Biotechnology) at room temperature for 5 min. After incubation with 0.3% H2O2 in PBS (Sigma-Aldrich; Merck KGaA) at room temperature for 20 min, 50 µl biotin-dUTP was added to label the samples for 1 h at 37˚C in the dark. To develop the colors, the samples were then incubated with 50 µl streptavidin-HRP for 30 min at 37˚C, followed by incubation in 0.5 ml DAB solution for another 30 min at 37˚C and the anti-fluorescence quencher was added dropwise for mounting. The coloration was observed microscopically from three random fields of view using a fluorescence microscope (magnification, x200; Olympus Corporation).
4
OSCC Cell Proliferation Assay
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After abovementioned transfection, OSCC cell proliferation was evaluated using colony formation assay. Cal27 and SCC9 cells were grown in the culture plate (3 × 103/100 µl) in an incubator for another 24 h at 37 °C containing 5% CO2. Every two days, the culture medium was changed. After 14 days, the incubation was terminated. Cells were purified twice with phosphate buffered saline (PBS) (Suntip, Guangdong, China) after removing the solution. Colonies were fixed with 5% paraformaldehyde (Macklin, Shanghai, China) and then stained with 1 ml of 0.1% crystal violet solution (Le Heng Technology, Shenyang, China). After PBS-washing, the colony number was counted using a gel documentation system (Thermo Fisher Scientific).
5
Alcian Blue Staining of Extracellular Matrix
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The cells on days 3, 7, 14, and 21 were fixed in 4% paraformaldehyde (Macklin, China) for 12 h at 4°C and stained with 1% (w/v) Alcian Blue 8GX (Solarbio, China) aqueous solution (Challa et al., 2010 (link)). The stained cells were assembled on plates, washed three times with deionized water to ensure that only specific staining remained, and photographed using a stereomicroscope (Jiangnan, China), and the relative staining level was processed using ImageJ software.
6
Osteoclast Differentiation and Assay
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RAW264.7 cells and BMMs were seeded in 96-well plates at 5 × 103 cells/well. After 24 h, osteoclasts were cultured in complete α-MEM containing RANKL (50 ng/ml) and administered various concentrations of DHT for 6–7 days.
Media change was performed every 2 days and the osteoclasts were scoured three times with PBS until the formation of mature osteoclasts. Thereafter, the cells were fixed with 4% paraformaldehyde (Macklin, Shanghai, China) for 3–5 min, stained according to the TRAcP kit procedure, and photographed using a standard inverted microscope (Olympus, Japan). Mature osteoclasts were observed when more than three nuclei were present in the stained multinucleated cells.
Media change was performed every 2 days and the osteoclasts were scoured three times with PBS until the formation of mature osteoclasts. Thereafter, the cells were fixed with 4% paraformaldehyde (Macklin, Shanghai, China) for 3–5 min, stained according to the TRAcP kit procedure, and photographed using a standard inverted microscope (Olympus, Japan). Mature osteoclasts were observed when more than three nuclei were present in the stained multinucleated cells.
7
Rapid Pyrolysis of Poplar Sawdust
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Bio-oil was produced from rapid pyrolysis of Poplar Sawdust at 500 °C. Urea, Phenol and Paraformaldehyde were analytical grade and purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China).
8
Osteoblast Lineage Evaluation via 5hmC
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The tibias were dissected and fixed in 4% paraformaldehyde (Macklin) at 4°C for 12 h, and incubated in 15% EDTA (EDTA, FREE ACID, Cat#E8040, Solarbio) for decalcification. The specimens were embedded in OCT and sectioned at 10 μm, then permeabilized with 0.3% Triton for 5 min. After that, the specimens were blocked in 1:10 goat serum for 1 h and then stained at 4°C overnight with primary antibody OSX (sc-393325, Santa Cruz Biotechnology, Mouse monoclonal, 1:100), or anti-5hmC (Active Motif, 39,769, 1:300). Goat anti-mouse IgG H&L Alexa 488 (ab150113, Abcam, 1:100) was used as the second antibody. DAPI (Sigma) was used for nuclear staining.
9
Immunofluorescence Staining of COL3
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We treated the cells as required by the experiment, then fixed with 4% paraformaldehyde (MACKLIN, Shanghai, China). Next, we blocked with 5% BSA (WOLSEN, Shenzhen, China) in PBS for 1 h at room temperature. Then, the cells were incubated with rabbit polyclonal anti-COL3 primary antibody (Wanlei, Shenyang, China) at a dilution of 1:50 overnight, followed by incubation with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody (Boster, Wuhan, China) for 1 h at room temperature; we used DAPI for nuclear staining. Finally, we used a fluorescence microscope to observe and photograph the cells.
10
Fast Pyrolysis of Larix gmelinii Biochar
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Bio-oil, obtained in a fluidized bed at 550 °C via the fast pyrolysis of Larix gmelinii (Rupr.) Kuzen, was made at the Lab of Fast Pyrolysis of Biomass and Productive Utilization (Beijing Forestry University, Beijing, China). Bio-oil is a complex mixture which contains water (30 wt %) and a broad range of organic compounds, including 33.42% Phenols, 29.56% ketones, 12.45% aldehydes, 10.05% polysaccharides, 9.33% organic acids, and 4.39% other compounds, such as esters and ethers. Phenol, paraformaldehyde, p-toluenesulfonic acid monohydrate and montmorillonite K-10 were purchased from Shanghai Macklin Biochemical Co., Ltd., Shanghai, China. Sodium hydroxide (NaOH), petroleum ether (30–60), and phosphoric acid were provided by Beijing Chemical Industries Reagent Co., Ltd., Beijing, China. Tween-80 was supplied by Guanghua Chemical Reagent Co., Ltd., Guangdong, China.
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