Hello Hanni Grace F

Autofluorescence can be a problem as it could interfere with detection of specific fluorescent signals, especially when the signal of interest is very dim. Most autofluorescence is detected at shorter light wavelengths with most absorbing in UV to Blue range (355-488 nm) and emitting in the Blue to Green range (350-550 nm). Autofluorescence can therefore be a problem in these light ranges as the signal to noise ratio is decreased resulting in reduced sensitivity and false positives.

There are several ways you could overcome autofluorescence.

1. As there is less autofluorescence at longer light wavelengths, fluorophores which emit above 600 nm will have less autofluorescence interference. The use of a very bright fluorophore will also reduce the impact of autofluorescence. So, choosing a fluorophore with emission spectra in the red and far-red regions will help distinguish specific staining from autofluorescence.

2. To lower tissue autofluorescence you can also treat the tissue with solutions of Sudan Black or similar non-fluorescent diazo dyes. These hydrophobic dye molecules will generally bind non-specifically to tissue sections. After binding to the tissue, Sudan Black acts as a mask to lower the fluorescence through the absorption of incident radiation (dark quenching).

3. Another method to diminish tissue autofluorescence is photobleaching. When this technique is used, tissue sections are exposed to high-intensity UV radiation for long periods of time to irreversibly photo-oxidize the fluorescent tissue elements. Photobleaching, which is often used in conjunction with other treatments, has been shown to be somewhat effective. However, it is time consuming.

4. You can also use the vector true view autofluorescence quenching kit which involves the treatment of tissue sections with an aqueous solution of a hydrophilic molecule that binds electrostatically to collagen, elastin, and RBCs. This non-fluorescent negatively charged molecule also binds effectively to formalin-fixed tissue including colon, pancreas, prostate, tonsil, spleen, kidney, gallbladder, and thymus.

Good Luck.