Fluorescent Dye
This compound targets and stains the enzymatic activity of tartrate-resistant acid phosphatase (TRAP), a key marker for osteoclasts, and alkaline phosphatase (ALP), a marker for osteoblasts. As a staining dye, it forms colored insoluble precipitates at sites of enzyme activity, enabling visual detection.

In situ bone osteoclast numbers[1]
Quantitation of osteoclasts in demineralized bone sections was based on previously described procedures. Fixed, demineralized, and paraffin embedded tibiae were sectioned and subjected to staining for tartrate resistant acid phosphatase (TRAP) with 0.1 mg/ml naphthol AS-MX phosphate and 0.6 mg/ml fast red violet LB salt in 0.1 M sodium acetate buffer, pH 5.0, containing 50mM sodium tartrate. Osteoclasts were identified as TRAP positive cells on the trabecular bone surfaces. The numbers of osteoclasts on the sections were counted and the fractions of bone surface occupied by osteoclasts were measured in the proximal tibia trabeculae using a color camera microscopy imaging system and the software of ImageJ (1.35s, NIH).

---

 Osteoclast potential[1]
The bone marrow cells were cultured (1.0 × 105 cells/0.5ml per well in a 48-well plate) for 6 days in α-MEM containing 10% FBS, 100U/ml penicillin, and 100μg/ml streptomycin. Cultures were fed every 3 days with medium containing rmM-CSF (20 ng/ml), rhsRANKL (60 ng/ml) and maintained at 37°C in a humidified atmosphere of 5% CO2. On day 6, cells were fixed in 10% formalin and stained for TRAP with 0.1 mg/ml naphthol AS-MX phosphate and 0.6 mg/ml fast red violet LB salt in 0.1 M sodium acetate buffer, pH 5.0, containing 50mM sodium tartrate. TRAP-positive cells with three or more nuclei were counted using phase-contrast microscopy.

---

As a staining reagent, Fast Red Violet LB is not typically studied for independent in vitro activity. It acts as a chromogenic substrate for phosphatases; when cleaved by TRAP or ALP, it produces a red-violet precipitate that localizes to cells expressing these enzymes, allowing visualization under light microscopy.

The dye is used to stain TRAP and ALP activity in tissue sections and cell cultures. It is widely applied in bone biology research to identify osteoclasts (TRAP-positive) and osteoblasts (ALP-positive), enabling quantification of bone resorption and formation activity in bone remodeling studies.

For ALP staining: fix cells in 4% paraformaldehyde for 10 min at 4degC. Prepare working solution: 0.1% naphthol AS-MX phosphate and 0.1% Fast Red Violet LB zinc chloride in 56 mM 2-amino-2-methyl-1,3-propanediol buffer. Incubate fixed cells in this solution for 10-15 min at room temperature in the dark. Wash with PBS and observe under light microscopy. For TRAP staining, add 50 mM sodium tartrate to the buffer to inhibit other phosphatases.

Cell viability and differentiation assay[2]
The viability of the cryopreserved cells was assayed using a NucleoCounter, an instrument for counting mammalian cells, which employs a fluorescence microscope adapted to a relatively low optical magnification. The cell samples were analyzed on the NucleoCounter before and after treatment with a lysis buffer, giving an estimate of nonviable and total cells.
To monitor osteoblastic differentiation, the cryopreserved cells were thawed in α-MEM containing 15% FBS. They were then seeded and cultured at a density of 1 × 104 cells/cm2 in a twelve-well culture plate in the medium supplemented with 10 mM β-glycerophosphate disodium salt, 0.07 mM L-ascorbic acid phosphate magnesium salt n-hydrate and 0.1 mM dexamethasone for two weeks. The cultures without dexamethasone were used as a negative control. Differentiated osteoblasts were biochemically analyzed by the determination of calcium and ALP activity staining. Calcium deposition (in vitro bone formation) was evaluated by the method we previously reported). Briefly, 1 µg/mL calcein was added to the medium during the culture period and the fluorescence of the incorporated calcein in the extracellular regions of the cells was observed by using a fluorescent microscope. The medium containing the calcein was removed and washed with PBS prior to observation. For alkaline phosphatase (ALP) activity staining, the cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min at 4°C. The fixed cells were then soaked in 0.1% naphthol AS-MX phosphate and 0.1% fast red violet LB salt in 56 mM 2-amino-2-methyl-1, 3-propanediol for 10 min at room temperature. Following a washing step with PBS, the active ALP cells were observed by microscopy[2].

---

Culture osteoclast or osteoblast cell lines or primary bone marrow-derived cells in appropriate differentiation media. After differentiation induction, remove culture medium and fix cells in 4% paraformaldehyde for 10 minutes at 4degC. Wash with PBS. Incubate with Fast Red Violet LB staining solution (prepared as described) for 10-30 minutes at room temperature. Wash thoroughly with PBS and air-dry. Observe red-violet staining under brightfield microscopy and quantify positive cell numbers or staining intensity.

Animal models are not applicable for direct staining protocols. However, for in situ staining, harvest bone tissue from mice or rats (e.g., femur or tibia), fix in 4% paraformaldehyde for 24-48 hours, decalcify in EDTA or formic acid solution for 7-14 days, and embed in paraffin or OCT. Cut sections (5-10 um), dewax and rehydrate, then incubate with Fast Red Violet LB staining solution as described above for visualization of osteoclast and osteoblast activities.

Fast Red Violet LB is applied ex vivo or in vitro for tissue staining and is not studied for systemic pharmacokinetic properties. When used as a histochemical dye, it remains localized to the tissue section or cultured cells, forming water-insoluble precipitates that do not diffuse significantly.

Fast Red Violet LB is considered low in toxicity as a histochemical reagent when used according to standard laboratory protocols. Inhalation, ingestion, or skin contact should be avoided. The compound should be handled with appropriate personal protective equipment (gloves, lab coat, safety glasses). Dispose of staining solutions according to institutional hazardous waste guidelines.

[1]. Genetic background influences fluoride's effects on osteoclastogenesis. Bone. 2007 Dec;41(6):1036-44.

[2]. Cultured autologous human cells for hard tissue regeneration: preparation and characterization of mesenchymal stem cells from bone marrow. Artif Organs. 2004 Jan;28(1):33-9.

Fast Red Violet LB is commonly used in combination with naphthol AS-MX phosphate as the substrate for phosphatase detection. The red-violet azo dye precipitate is stable and does not fade quickly under light microscopy. It is part of the standard TRAP staining kit widely used for osteoclast identification in bone histomorphometry. Alternative names include Fast Red Violet LB salt.

C14H11CLN3O.1/2ZNCL2

340.86

Fast Red Violet LB;32348-81-5

Off-white to yellow solid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~62.5 mg/mL (~183.36 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)