Naphthol as bi alkaline solution

Manufactured by Merck Group

Naphthol AS-BI alkaline solution is a laboratory reagent used in histological and cytological applications. It is a pre-prepared solution containing the compound Naphthol AS-BI, which is commonly used as a substrate in enzyme histochemistry procedures.

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Naphthol AS-BI

6 protocols using naphthol as bi alkaline solution

ALP Activity Assay Protocol

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After 7 and 14 days, cell layers were fixed with citrate-acetone-formaldehyde fixative solution for 1 min followed by three washes with distilled water. An alkaline dye consisting of a diazonium salt solution and naphthol AS-BI alkaline solution (Procedure No. 86, AP, leukocyte; Sigma Aldrich) was added to the cell layer and incubated in reduced light conditions at room temperature for 15 min. The monolayers were washed again with distilled water, and the cell layers were then counterstained with neutral red solution for 5 min. Cells exhibiting ALP activity were marked by blue staining. Representative pictures of the stained monolayers were obtained by microscopy and digital photography.

Andrietti A.L., Durgam S.S., Naumann B, & Stewart M. (2023). Basal and inducible Osterix expression reflect equine mesenchymal progenitor cell osteogenic capacity. Frontiers in Veterinary Science, 10, 1125893.

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Alkaline Phosphatase Activity Assay

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Fixative solution and ALP staining solution are required for the ALP assay. Fixative solution is a mixture of 25 ml citrate solution (Sigma), 65 ml acetone (Junsei Chemical, Tokyo, Japan) and 8 ml 37 % formaldehyde. To create the ALP staining solution, 1 ml sodium nitrite solution (Sigma) was mixed with 1 ml FBV solution (Sigma). After incubation at room temperature for 2 min, 45 ml DW and 1 ml naphthol As-BI alkaline solution (Sigma) were added to the mixture. For the ALP assay, the cells were fixed in fixative for 30 s and then incubated in ALP staining solution for 20 min in the dark.

Kim D., Choi J., Han K.M., Lee B.H., Choi J.H., Yoo H.W, & Han Y.M. (2015). Impaired osteogenesis in Menkes disease-derived induced pluripotent stem cells. Stem Cell Research & Therapy, 6(1), 160.

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MSC Osteogenesis Regulated by BMP-2 Signaling

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Purified rhBMP-2, IL-6, and sIL-6R were provided by R&D Systems (Minneapolis, MN, USA). Monensin, dorsomorphin homolog 1 (DMH1), naphthol AS-BI alkaline solution, and phalloidin were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). Rabbit anti-Smad1 antibody, rabbit anti-BMPR1A antibody, mouse anti-BMPR2 antibody, rabbit anti-CCAAT enhancer-binding protein-α (C/EBPα) antibody and rabbit anti-peroxisome proliferator-activated receptor gamma (PPARγ) antibody were obtained from Abcam (Cambridge, MA, USA). Anti-phospho-Smad1/5/8 antibody, anti-phospho-p38 antibody, anti-p38 antibody, anti-Runx2 antibody, anti-GAPDH antibody, anti-β-actin antibody, and anti-fade reagent were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit anti-BMPR1B antibody, Alexa Fluor-488-conjugated secondary antibody, and TRIzol reagent were obtained from Invitrogen (Carlsbad, CA, USA). FuGENE®HD transfection reagent was provided by Promega BioSystems (Sunnyvale, CA, USA). Smad1 shRNA plasmids were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Human MSC osteogenic and ADM were purchased from Cyagen Biosciece (Guangzhou, China). Lewis rats were provided by Shanghai Experimental Animal Center China.

Huang R.L., Sun Y., Ho C.K., Liu K., Tang Q.Q., Xie Y, & Li Q. (2018). IL-6 potentiates BMP-2-induced osteogenesis and adipogenesis via two different BMPR1A-mediated pathways. Cell Death & Disease, 9(2), 144.

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Alkaline Phosphatase Assay of CFU-F

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Marrow cells were harvested from six 4-week-old TSP2-rep mice, pooled, and plated in a 60 mm plate at a density of 4 million cells per plate and the experiment was repeated four times. Cells were grown for 14 days in culture media (Alpha-MEM, 10% FBS, 1%, L-glutamine, 1% anti-anti) in a humidified condition of 5% CO₂ at 37°C. A half exchange of culture media was performed every third day. Whole plates were imaged for T2GFP expression every other day. On day 14 after T2GFP imaging, plates were stained for alkaline phosphatase (ALP) and imaged using brightfield microscopy.
Alkaline phosphatase staining was performed on the CFU-F plates as a marker of preosteoblasts. Plates were washed twice with PBS and then fixed with citrate-acetone-36% formaldehyde (ratio, 25:65:8) for 30 seconds and then rinsed with deionized water. Naphthol AS-BI alkaline solution (861–10; Sigma-Aldrich) was added to the plates at 25°C for 15 minutes and then rinsed with deionized water. Plates were counterstained at 25°C for 2 minutes with Neutral Red (N6264; Sigma-Aldrich). Finally, plates were rinsed with tap water and allowed to air dry.

Zondervan R.L., Jenkins D.C., Reicha J.D, & Hankenson K.D. (2020). Thrombospondin-2 spatiotemporal expression in skeletal fractures. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 39(1), 30-41.

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Histochemical Evaluation of ALP Activity

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After exposure to osteogenic medium for 1 week, cells were washed with PBS and fixed with citrate‐acetone‐formaldehyde fixative and washed again three times with PBS. Extracellular ALP activity was examined histochemically using Naphthol AS‐BI alkaline solution as per manufacturer's protocol (Sigma, Irvine, U.K.). After ALP staining, the samples were washed with PBS and imaged.

Thiagarajan L., Abu‐Awwad H.A, & Dixon J.E. (2017). Osteogenic Programming of Human Mesenchymal Stem Cells with Highly Efficient Intracellular Delivery of RUNX2. Stem Cells Translational Medicine, 6(12), 2146-2159.

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Alkaline Phosphatase Cell Staining

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Cells were washed once with PBS and fixed in 3.7% formaldehyde for 5 min at room temperature. Cells were then incubated with AP staining buffer (FRV-Alkaline solution/sodium nitrite solution/naphthol AS-BI alkaline solution; Sigma) at room temperature for 10–30 min. Images of the stained cells were captured with a Nikon (ECLIPSE Ti-U) reverse phase-contrast microscope.

He H., Wang C., Dai Q., Li F., Bergholz J., Li Z., Li Q, & Xiao Z.X. (2016). p53 and p73 Regulate Apoptosis but Not Cell-Cycle Progression in Mouse Embryonic Stem Cells upon DNA Damage and Differentiation. Stem Cell Reports, 7(6), 1087-1098.

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