5(6)-FITC (Fluorescein isothiocyanate), or Fluorescein 5(6)-isothiocyanate, is a versatile amine-reactive derivative of fluorescein dye. It serves as an effective label for antibodies and other probes, finding utility in fluorescence microscopy, flow cytometry, and immunofluorescence-based assays such as ELISA and Western blotting.

Instructions
1. Solvent selection:
5(6)-FITC is dissolved in anhydrous dimethyl sulfoxide (DMSO) at a typical concentration of 1–10 mg/mL. The solution can be diluted to achieve the desired labeling concentration.
2. Labeling reaction:
1) Add 5(6)-FITC solution to a solution containing the target molecule (such as an antibody or protein), usually using a 5-10-fold molar ratio of FITC.
The reaction is usually carried out at 4°C for 1-2 hours without light exposure.
2) Dilute FITC with PBS or other suitable buffer and adjust the pH to around 8.0 to promote the reaction.
3. Reaction termination:
1) After the labeling reaction, unreacted FITC needs to be removed by dialysis or gel filtration column.
2) The labeled sample can be used immediately for downstream experiments or frozen for storage.
4. Detection and analysis:
Fluorescence microscopy: Observe the labeled samples with a fluorescence microscope. The excitation wavelength of FITC is 495 nm and the emission wavelength is 519 nm.
Flow cytometry: Analyze the fluorescence signal by flow cytometry. The green fluorescence emitted by FITC can be used to quantitatively analyze cell labeling.
Fluorescence spectrophotometer: Detect the fluorescence intensity of the sample, usually at 495 nm excitation and 519 nm emission.