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 immunohistochemical (IH) studies of this actin-based motor. We show here that BM-V immunoreactivity (IR) can be retrieved from formalin-fixed, paraffin-embedded human tissue. BM-V IR was optimally retrieved by boiling 5 microm cerebellar tissue sections in 10 mM sodium citrate buffer, pH 6, for 15 min, using a microwave oven set at 900 W and 2.45 GHz. A polyclonal, affinity purified anti-BM-V antibody, raised in rabbits against the tail domain of chicken BM-V, was shown here to recognize a single band in Western blots of human 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 in granule cells. The same pattern of BM-V IR expression was consistently and maximally detected in tissues stored in 10% formalin from 1 week to 2.5 months. The AR protocol for BM-V described here permits its IH study in formaldehyde-fixed tissues. It is a valuable tool to study BM-V in well fixed tissues, as occurs with the large collection of human archival tissue available.
Martins
A R
AR
Department of Pharmacology, Faculty of Medicine of Ribeirão Prêto, University of São Paulo, Brazil. armartin@fmrp.usp.br
Dias
M M
MM
Vasconcelos
T M
TM
Caldo
H
H
Costa
M C
MC
Chimelli
L
L
Larson
R E
RE
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification*; D002531:Cerebellum / Q000737:chemistry*; D002645:Chickens; D002675:Child, Preschool; D005333:Fetus; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007223:Infant; D008872:Microwaves; D024701:Myosin Type V; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D016612:Paraffin Embedding; D011817:Rabbits; D013702:Temporal Lobe / Q000737:chemistry*
D002148:Calmodulin-Binding Proteins; D009419:Nerve Tissue Proteins; C078829:chicken brain myosin-V p190; D005557:Formaldehyde; D024701:Myosin Type V