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.