Elimination of artifactual immunogold labeling from secondary walls of woody stem sections

Abstract

Preliminary in situ transmission electron microscopy immunogold localization of a 24-kDa dehydrin-like protein in red-osier dogwood (Cornus sericea L.) stem cross-sections was contaminated with extensive background labeling of secondary cell walls in both positive and negative control samples. Alterations in antibody dilution, buffer salt concentration and stringency of the washing solutions failed to eliminate background cell-wall labeling. A procedure was developed in which lyophilized cold-acclimated C. sericea xylem tissue was pulverized and boiled with sodium dodecyl sulfate (SDS)-protein extraction buffer to remove soluble proteins and to inactivate proteases. Wood powder, treated with SDS-protein extraction buffer, was used to pre-absorb chicken immune serum specific for a 24-kDa dehydrin-like protein prior to immunolocalization assays. Pre-incubation of primary antibodies did not compromise the recognition of the 24-kDa protein, and this technique effectively eliminated background cell-wall labeling.