Selective visualisation of aromatic and aliphatic suberin domains using combined Sudan Red-7B staining and UV autofluorescence.

This article reports a visualization method that combines Sudan red 7B staining with UV autofluorescence in rice root secondary cell walls. Sudan red 7B stains the aliphatic domains of suberin (SPAD), as well as intracellular lipids, whereas after UV excitation of unstained sections the aromatic domains of suberin (SPPD) emitted violet autofluorescence. However, when Sudan red 7B staining preceded UV excitation, the suberized cell walls fluoresced in varying colors from red to magenta, violet or bluish white. The optical phenomenon, Inner Filter Effect (IFE), exhibited this color variation as the differential solubility of Sudan Red 7B stain-SPAD suberin domain complex acted as an optical filter that shifted the violet autofluorescence from SPPD suberin domains to red. At the time of visualization, the red fluorescence spectrally mixed with violet autofluorescence such that the suberized cell walls fluoresced in varying colors. Using this visualization procedure, the intramolecular deposition and distribution pattern for SPAD and SPPD suberin domains in the outer apoplastic barrier can be effectively ascertained.