Rhodamine immunohistofluorescence applied to plant tissue

Area of Science:

• Plant Biology
• Biochemistry
• Microscopy

Background:

• Autofluorescence in plant tissues can interfere with immunohistochemical staining.
• Sanifoin (Onobrychis viciifolia, Scop.) root and seed tissues exhibit strong autofluorescence under blue light (495 nm).
• This autofluorescence is spectrally similar to fluorescein, a common immunohistochemical fluorochrome.

Purpose of the Study:

• To evaluate the utility of rhodamine isothiocyanate for immunohistochemical localization in plant tissues.
• To identify a fluorochrome that minimizes interference from plant autofluorescence.

Main Methods:

• Excitation of sanifoin tissue with blue light (495 nm) to observe autofluorescence.
• Coupling rhodamine isothiocyanate to immunoglobulin.
• Excitation of rhodamine-coupled immunoglobulin with green light (546 nm) and observation with a 580 nm barrier filter.

Main Results:

• Sanifoin root and seed tissues show bright autofluorescence under blue light, comparable to fluorescein.
• Rhodamine isothiocyanate exhibits reddish-orange fluorescence (emission max 590 nm) when excited at 546 nm.
• Plant tissues display minimal autofluorescence when excited at 546 nm and viewed with a 580 nm barrier filter.

Conclusions:

• Rhodamine isothiocyanate is a suitable fluorochrome for immunohistochemical staining in plant tissues.
• Utilizing rhodamine with green light excitation (546 nm) effectively circumvents autofluorescence issues in plant samples.
• This approach enhances the reliability and interpretability of immunohistochemical staining in plant research.