Unconventional antigen retrieval for carbohydrate and protein antigens

Aldehyde fixation of tissues often adversely affects the reactivity of cellular proteins with antibodies. A most commonly used retrieval technique in immunohistochemistry is high-temperature microwave heating of sections from formaldehyde-fixed and paraffin-embedded tissues. Here we report that pretreatment of paraffin and ultrathin cryosections with N-glycanase F to remove N-glycosidically linked oligosaccharides can result in a dramatic increase in specificity and intensity of immunogold labeling for sugar moieties present on O-glycosidically linked oligosaccharides. This is demonstrated in the immunolocalization of poly alpha2,8 KDN (KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) of megalin in rat kidney. The mechanism of this retrieval procedure is most probably based on the elimination of sterical hindrance by large N-glycosidically linked oligosaccharides. Furthermore, we demonstrate that exposure of ultrathin cryosections to acidic conditions (pH 5.5) at ambient temperature prior to immunogold labeling can result in an increased labeling intensity. This effect was observed for megalin immunoreactive sites in proximal tubular epithelia of rat kidney. It is proposed that the mechanism of this retrieval procedure is based on the depolymerization of methylen and polymethylen bridges introduced by formaldehyde in the acidic milieu.