6-Carboxyfluorescein (6-FAM) contains a carboxylic acid functional group, facilitating reactions with primary amines through carbodiimide activation.

6-FAM labeled oligonucleotide probes for fluorescence in situ hybridization (FISH)
I. Solution preparation:
1. Preparation of stock solution: Dissolve 6-FAM in DMSO or ethanol to prepare a 1-10 mM stock solution; (it is recommended to store at -20 ℃ or -80 ℃ in the dark after aliquoting)
2. Preparation of working solution: Dilute the 6-FAM stock solution with PBS or other suitable buffer, usually 1-10 µM; (Select the appropriate working solution concentration according to experimental requirements, and prepare it for immediate use)
II. Operation steps:
1. Sample preparation: Prepare chromosome samples of chicken embryonic cells, including colchicine treatment, hypotonic swelling and methanol-glacial acetic acid fixation.
2. Pretreatment: Chromosomes are treated with RNase A, pepsin and formaldehyde.
3. Hybridization: 6-FAM labeled oligonucleotides were dissolved as probes in hybridization buffer (50% formamide, 2×SSC, 10% dextran sulfate) containing E. coli tRNA at a concentration of 20 ng/μL. After co-denaturation of DNA and probe, hybridization was performed overnight at room temperature in a humidified box;
4. Washing and counterstaining: After hybridization, the slides were washed three times in 2×SSC, dehydrated in a series of 70%-96% ethanol, and sealed with an antifade containing DAP I after drying;
5. Observation and analysis: The samples were observed using a Leica DM4000B fluorescence microscope equipped with a monochrome CCD camera DFC350FX and appropriate filters, and the chromosome color images were captured and processed using QFISH software.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.

DMSO: 40 mg/mL (106.29 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween-80+45% Saline: 2 mg/mL (5.31 mM), Sonication is recommended.