Microwave-stimulated recovery of myosin-V immunoreactivity from formalin-fixed, paraffin-embedded human CNS

The lability of brain myosin-V (BM-V) to aldehyde-fixation has hindered imm unohistochemical (IH) studies of this actin-based motor. We show here that BM-V immunoreactivity (IR) can be retrieved from formalin-fixed, paraffin-e mbedded human tissue. BM-V IR was optimally retrieved by boiling 5 mu m cer ebellar tissue sections in 10 mM sodium citrate buffer, pH 6, for 15 min, u sing a microwave oven set at 900 W and 2.45 GHz. A polyclonal, affinity pur ified anti-BM-V antibody, raised in rabbits against the tail domain of chic ken BM-V, was shown here to recognize a single band in Western blots of hum an cortical homogenates. The combined use of this monospecific antibody and of the antigen retrieval (AR) method above allowed us to verify that BM-V IR is strongly expressed in human Purkinje cell bodies and dendrites, and i n granule cells. The same pattern of BM-V IR expression was consistently an d maximally detected in tissues stored in 10% formalin from 1 week to 2.5 m onths. The AR protocol for BM-V described here permits its IH study in form aldehyde-fixed tissues. It is a valuable tool to study BM-V in well fixed t issues, as occurs with the large collection of human archival tissue availa ble. (C) 1999 Elsevier Science B.V. All rights reserved.