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| Product Name | DAPI Mounting Medium, Antifade, for Nuclear Counterstaining |
| Catalog No. | ATRSA-0032 |
| Description | Ready-to-use aqueous mounting medium containing DAPI (4',6-diamidino-2-phenylindole) and proprietary antifade agents for simultaneous nuclear counterstaining and coverslipping of immunofluorescence and FISH (fluorescence in situ hybridization) samples. DAPI is a cell-permeant, DNA-specific fluorochrome that binds selectively to adenine-thymine (A-T) rich regions of the minor groove of double-stranded DNA with approximately 20-fold fluorescence enhancement upon binding (Ex/Em 358/461 nm). The antifade formulation (containing PPD, DABCO, or proprietary compounds) retards photobleaching of DAPI and other fluorophores, preserving fluorescence signal during microscopy and image acquisition. |
| Intended Use | Nuclear counterstaining and mounting of immunofluorescence, FISH, and direct fluorescent labeling samples on glass slides for fluorescence microscopy; total cell count normalization in imaging-based assays; nuclear morphology assessment (apoptotic nuclear condensation/fragmentation). |
| Principle / Technology | DAPI intercalates into A-T rich regions of DNA minor groove; fluorescence enhancement upon DNA binding (quantum yield increase from 0.001 to ~0.3); blue emission (461 nm) upon UV excitation (358 nm); antifade agents reduce reactive oxygen species generation that causes fluorophore photobleaching. |
| Detection Method | Apply 1-2 drops of DAPI mounting medium directly onto stained specimen on glass slide; carefully lower coverslip avoiding air bubbles; allow to cure for 10-30 minutes at RT in dark; seal edges with nail polish for long-term storage; image using DAPI/UV filter set. |
| Sample Type | Immunofluorescence-labeled cells or tissue sections on glass slides; FISH preparations; TUNEL-stained sections; fluorescent protein-expressing fixed cells; cytospin preparations. |
| Performance Range / Specifications | DAPI nuclear staining: intense blue fluorescence of all nuclei; staining effective with DNA content from 0.1-10 pg per nucleus (haploid to polyploid); mounting medium refractive index ~1.45-1.50 matching glass and immersion oil. |
| Sensitivity / LOD | Detection of individual nuclei with DNA content as low as 0.1 pg (e.g., apoptotic bodies, micronuclei); nuclear counterstain detectable with exposure times of 10-100 ms on standard fluorescence microscope. |
| Specificity | DAPI is highly specific for double-stranded DNA; <5% fluorescence with single-stranded DNA or RNA (DAPI-RNA complex has shifted emission to ~500 nm); does not stain cytoplasm or extracellular matrix. |
| Reaction Conditions / Protocol | Coverslipping and staining occur simultaneously upon mounting; DAPI penetrates nuclei within seconds at room temperature. |
| Components / Formulation | DAPI (2 µg/mL final) in antifade mounting medium (glycerol/PVA or proprietary polymer-based), pH 8.0-8.5. |
| Storage Conditions | Store at 2-8 °C protected from light; do not freeze; warm to room temperature before use. |
| Shelf Life | 12 months from date of manufacture at 2-8 °C. |
| Package Specifications | 10 mL dropper bottle (sufficient for ~200-400 coverslips), 20 mL. |
| Product Form | Viscous liquid, colorless to pale yellow; fluorescent blue under UV light. |
| Quality Control | Each lot tested for DAPI staining intensity (standard HeLa cell nuclei), nuclear specificity (absence of cytoplasmic signal), antifade efficacy (fluorescence retention after 10-min continuous excitation vs. non-antifade control), and refractive index. |
| Key Features | Ready-to-use single-step mounting and counterstaining; DAPI with antifade protection; aqueous-based (no organic solvent); DAPI penetrates within seconds; compatible with multiple fluorophores; long-term signal preservation; nuclear morphology assessment. |
| Purity | DAPI ≥98% by HPLC; antifade components analytical grade. |
| Concentration | DAPI 2 µg/mL in mounting medium. |
| Activity / Unit Definition | DAPI-DNA binding: Kd ~10-9 M for A-T rich sites; fluorescence quantum yield ~0.3 when DNA-bound. |
| Molecular Weight | DAPI: 277.32 g/mol (C16H15N5 · 2HCl). |
| Source / Origin | Synthetic DAPI; glycerol-based or PVA-based aqueous mounting medium. |
| pH Range / Optimal pH | Mounting medium pH 8.0-8.5. |
| Shipping Conditions | Cold pack (2-8 °C); protect from light. |
| Expiration Date / Stability | 12 months at 2-8 °C; DAPI gradually loses fluorescence over time — use within 3 months after opening or if blue color fades. |
| Regulatory / Compliance | For research use only; DAPI is a potential mutagen — handle with appropriate PPE. |
| Compatibility | Compatible with most fluorophores including FITC/Alexa Fluor 488, TRITC/Cy3, Texas Red/Alexa Fluor 594, and Cy5/Alexa Fluor 647. Minimal spectral overlap with green and red fluorophores. Compatible with formalin-fixed, methanol-fixed, and acetone-fixed preparations. Thick specimens (>50 µm) may require longer mounting medium penetration time. |
| Recommended Buffer System | Tris-HCl or carbonate-based aqueous mounting medium, pH 8.0-8.5. |
| Application Notes / Precautions | Warm mounting medium to room temperature before use to reduce viscosity and avoid air bubbles during coverslipping. Allow mounted slides to cure in dark for at least 30 minutes (overnight for semi-permanent mounting). For long-term storage, seal coverslip edges with clear nail polish. For best antifade performance, store slides at 2-8 °C in the dark. DAPI intensity decreases over time even with antifade — image within 1 week for optimal signal. For DNA content analysis by densitometry, ensure uniform DAPI staining across the slide. |
| Batch-to-Batch Consistency | DAPI concentration within ±10% of specification; nuclear staining intensity within ±15% of reference lot; antifade efficacy within ±10%. |
For research use only, not for clinical use.
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