After deparaffinization, osteosarcoma tumor sections were treated with 0.3% H2O2 for 15 min at RT, washed and boiled in citrate buffer. Sections were blocked with 3% BSA/PBS for 60 min at RT and incubated with primary rat anti-mouse CD31 antibody (1:50; Dianova) and goat anti-human/mouse desmin antibody (1:200; R&D systems; AF3844) in 3.0% BSA/PBS overnight at 4°C. Primary antibody was detected with secondary rabbit anti-rat HRP (1:100; DakoCytomation; P045001) and rabbit anti-goat biotinylated (1:200; DakoCytomation) in 3.0% BSA/PBS for 30 min at RT. After a washing step, sections were developed with DAB substrate to detect the CD31 staining. To detect the pericytes, sections were washed and incubated with strep-AP (1:500; Genmed Synthesis Inc. San Antonio, Texas, USA) in 10X TBS Solution (0.5 M Tris-Cl; 1.5 M NaCl, pH 7.6) for 30 min at RT. Washed in ddH2O and developed with Fast Blue BB (Sigma Aldrich). Finally, sections were washed in ddH2O, air-dried and embedded in Kaisers glycerol gelatin (Merck group).
Fast blue bb
Manufactured by Merck Group
Sourced in United States, Germany
Fast Blue BB is a laboratory reagent used in staining and histochemical procedures. It is a diazonium salt that can be used to detect the presence of certain enzymes or compounds in biological samples. The core function of Fast Blue BB is to provide a colorimetric or fluorescent signal when reacting with the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Naphthol as bi phosphate, by Merck Group (6 mentions)
Naphthol as mx phosphate, by Merck Group (4 mentions)
Alizarin red, by Merck Group (4 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
β glycerophosphate, by Merck Group (2 mentions)
Naphthol as mx, by Merck Group (2 mentions)
Rat anti mouse cd31 antibody, by Dianova (1 mentions)
Rabbit anti rat hrp, by Agilent Technologies (1 mentions)
Kaiser s glycerol gelatin, by Merck Group (1 mentions)
P0321, by Beyotime (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Disodium p nitrophenyl phosphate, by Fujifilm (1 mentions)
Sh 1000, by Corona Electric (1 mentions)
L ascorbic acid 2 phosphate, by Fujifilm (1 mentions)
Alp kit, by Merck Group (1 mentions)
Potassium acetate, by Merck Group (1 mentions)
Acetone, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Thermo Fisher Scientific (1 mentions)
Xylenol orange disodium salt, by Merck Group (1 mentions)
Nitroblue tetrazolium chloride, by Merck Group (1 mentions)
24 protocols using fast blue bb
1
Dual Immunohistochemical Staining for Tumor Vasculature
2
In Vitro Osteogenic Differentiation of MSCs
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In vitro osteogenesis of MSCs was induced using the osteogenic differentiation medium, containing 50 μg/ml ascorbic acid, 5 mM β-glycerophosphate, 100 nM dexamethasone (all from Sigma)25 (link), supplemented with or without 2 mM αKG.
On day 10, cells were fixed with 4% polyoxymethylene for 30 min. After washing cells with PBS for three times, cells were incubated at 37 °C with 0.1 M Tris buffer (pH 9.3) consisting 0.25% naphthol AS-BI phosphate (#N2125, Sigma) and 0.75% Fast Blue BB (D9805, Sigma). Images of stained plates were obtained using a scanner (#V330, EPSON). Quantification of alkaline phosphatase (ALP) activity was performed using a commercial kit, according to the instructions (#P0321, Beyotime). And the optical density was examined via spectrophotometer (Thermo Fisher Scientific) at 405 nm.
On day 10, cells were fixed with 4% polyoxymethylene for 30 min. After washing cells with PBS for three times, cells were incubated at 37 °C with 0.1 M Tris buffer (pH 9.3) consisting 0.25% naphthol AS-BI phosphate (#N2125, Sigma) and 0.75% Fast Blue BB (D9805, Sigma). Images of stained plates were obtained using a scanner (#V330, EPSON). Quantification of alkaline phosphatase (ALP) activity was performed using a commercial kit, according to the instructions (#P0321, Beyotime). And the optical density was examined via spectrophotometer (Thermo Fisher Scientific) at 405 nm.
3
BMP-2 Induced Alkaline Phosphatase Activity
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Cells were plated in 96-well plates at a density of 1 × 104 cells/well, then cultured for 72 h in the above-mentioned medium containing BMP-2 (100 ng/ml), in the presence and absence of either 8-nitro-cGMP or 8-bromo-cGMP (30 μmol/L). Cells were fixed for 30 min in 4% paraformaldehyde and washed with PBS, then incubated for 30 min at 37°C with 100 mmol/L Tris-HCl buffer (pH 8.5) containing 270 μmol/L naphthol AS-MX phosphate (Sigma-Aldrich) and 1.4 mmol/L Fast blue BB (Sigma-Aldrich). After washing with tap water, they were observed under a microscope.
Cells were cultured in the same conditions described above, washed with PBS, and homogenized with 1% Nonidet P-40 (50 μl) under sonication on ice. Cell lysates (10 μl) were added to 50 μl of 0.2 mol/L Tris–HCl buffer (pH 9.5) containing one mmol/L MgCl2 and 12.5 mmol/L disodium p-nitrophenyl phosphate (Fujifilm Wako Pure Chemical Co.). After incubation for 15 min at 37°C, the reactions were terminated by adding 50 μl of 0.5 mol/L NaOH, and the absorbance of the reaction mixture at 405 nm was read using a microplate reader (SH-1000; Corona Electric, Ibaraki, Japan). The increase in absorbance after 15 min was divided by the amount of cellular protein, with the obtained value used to express the specific activity of ALP.
Cells were cultured in the same conditions described above, washed with PBS, and homogenized with 1% Nonidet P-40 (50 μl) under sonication on ice. Cell lysates (10 μl) were added to 50 μl of 0.2 mol/L Tris–HCl buffer (pH 9.5) containing one mmol/L MgCl2 and 12.5 mmol/L disodium p-nitrophenyl phosphate (Fujifilm Wako Pure Chemical Co.). After incubation for 15 min at 37°C, the reactions were terminated by adding 50 μl of 0.5 mol/L NaOH, and the absorbance of the reaction mixture at 405 nm was read using a microplate reader (SH-1000; Corona Electric, Ibaraki, Japan). The increase in absorbance after 15 min was divided by the amount of cellular protein, with the obtained value used to express the specific activity of ALP.
4
Osteogenic Differentiation Assay
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Cells were cultured in osteogenic medium containing 10 mM β-glycerophosphate (Sigma-Aldrich), 100 μM L-ascorbic acid 2-phosphate (Wako), and 100 nM dexamethasone (Sigma) for indicated time periods. For alkaline phosphatase staining, after 4 weeks of induction, cells were fixed with 4% paraformaldehyde for 1 min at room temperature and incubated with a solution of 0.25% naphthol AS-BI phosphate and 0.75% Fast Blue BB (Sigma-Aldrich, St Louis, MO, USA) dissolved in 0.1 M Tris buffer (pH 9.3). For Alizarin Red staining to detect mineralized nodule formation, cells were fixed cells for 30 min at room temperature and incubated with 2% Alizarin Red (Sigma-Aldrich, St Louis, MO, USA).
5
Osteogenic Differentiation Visualization
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The cells were fixed with 4% paraformaldehyde for 10 min after 10 days induction of osteogenic differentiation. Then, the samples were incubated in 0.1 M Tris buffer (pH 9.3) containing 0.25% naphthol AS-BI phosphate (Sigma-Aldrich, Saint Louis, MO, USA) and 0.75% Fast Blue BB (Sigma-Aldrich) at 37 °C.
6
Osteogenic Differentiation Quantification
7
Comprehensive Biochemical Assay Protocols
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Phosphate phosphate, sodium Chloride, acetone, sodium carbonate were purchased from Acros Organics (USA). Methanol (MeOH), Dithiothreitol (DTT), Ethyline Diamine Tetra Acetic acid (EDTA), 2-propanol, Nitro blue tetrazolium chloride (NBT), Guaiacol, α-Naphthyl acetate solution, Fast blue BB, xylenol orange disodium salt, sulfuric acid (H2SO4), sodium Chloride (NaCl), ferrous ammonium sulphate, glycerol, o-Dianisidine, Potassium acetate, α-amylase enzyme, 3, 5-dinitrosalicylic acid, bovine serum albumin, hydrochloric acid (HCl), hydrogen peroxide H2O2, sodium acetate, Glacial acetic acid, Aluminium chloride AlCl2, sodium phosphate, and 2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTs) were purchased from Sigma-Aldrich (Merck Germany). Polyvinyl Pyrrolidone (PVPP), starch and Acetic acid were purchased from Bio-world GeneLinx International, Inc., USA. Folin-Ciocalteu (FC) Reagent, Bradford dye, Sodium Dodecyl Sulfate (SDS) were purchased from Thermo Fisher Scientific UK. 2, 6-dichloroindophenol (DCIP) was purchased from Millipore Sigma US.
8
Alkaline Phosphatase Activity Assay
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Cells were plated in 96-well plates at a density of 1 × 104 cells/well, and cultured for 72 hours in the presence or absence of BMP-2 (300 ng/mL). Cells were fixed for 30 minutes in 4% paraformaldehyde and washed with PBS, then incubated for 30 minutes at 37 °C with 100 mmol/L Tris-HCl buffer (pH 8.5) containing 270 μmol/L naphthol AS-MX phosphate (Sigma-Aldrich) and 1.4 mmol/L Fast blue BB (Sigma-Aldrich). After washing with tap water, they were observed under a microscope.
9
Alkaline Phosphatase Activity Staining
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Cells were stained for alkaline phosphatase activity. Briefly, cells were fixed in 2% PFA/0.2% gluteraldehyde for 1h, and then incubated with substrate solution for 30 min at 37°C. Substrate solution contained 8 mg napthol AS-TR (Sigma) in 0.3 mL n-n'dimethylformamide (Sigma) mixed with 24 mg fast blue BB (Sigma) in 30 mL 100 mM Tris-HCL (pH 9.6). Subsequently, 10 mg MgCL2 was added, the pH was adjusted to 9.0, and then the entire solution was filtered (0.2 μM pore size).
10
Osteoblast Differentiation Assay Protocol
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To culture osteoblasts, calvarias of neonatal mice were digested with 0.1% collagenase and 0.2% dispase 5 times, and cells isolated in the last 3 digestions were combined and cultured in α-MEM containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. To measure alkaline phosphatase (ALP) activity, primary osteoblasts were inoculated at a density of 2 × 104 cells/well and cultured in the absence or presence of 50 μg/mL ascorbic acid and 10 mM β-glycerol phosphate. On differentiation day 7, cells were sonicated in 50 mM Tris–HCl buffer (pH 7.4) containing 1% Triton X-100, 150 mM NaCl, and 1 mM EDTA. Then, 100 μL of substrate (p-nitrophenylphosphate) (Sigma) was added to the cells, and the plate was incubated for 30 min at 37 °C. The amount of p-nitrophenol released was determined by measuring absorbance at 405 nm using a microplate reader. For ALP staining, cells were fixed in 70% ethanol and stained for 10–20 min with a solution containing 0.01% naphthol, AS-MX phosphate, 1% N,N-dimethyl formamide, and 0.06% fast blue BB (Sigma).
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