Analysis of the water channel protein AQP4 by a modified and
highly sensitive postembedding immunogold procedure
E. A. Nagelhus, F.-M. Haug, O.P. Gttersen
Department of Anatomy, University of Oslo, P.O.B. 1105 Blindem, 03 17 Oslo, Norway
Postembedding
immunogold cytochemistry is often the method of choice for high
resolution localization of membrane proteins. However, with this approach, labelling is restricted to
those epitopes that are expressed at the surface of the section. These constitute but a minor fraction
of the epitopes, implying that sensitivity becomes a critical factor. Here we report the results of our
efforts to increase the sensitivity of postembedding
immunogold analyses of the water channel
protein aquaporin-4 (AQP4). We also describe the use of an image analysis program for
semiautomatic evaluation of gold particle distributions along cell membranes. Our procedures have
proved useful in studies of several membrane proteins (in addition to AQP4), including glutamate
receptors and monocarboxylate transporters.
Aldehyde fixed specimens of rat brain and retina were freeze substituted [ 11, embedded in a
metachrylate resin (usually Lowicryl HM 20), sectioned at -50-80 nm, and processed according to a
two-step immunogold protocol [2]. The following modifications of the standard protocol were
found to enhance the sensitivity of the AQP4 immunolabelling:
1, immersion or perfusion fixation
by a “pH-shift method” (4% formaldehyde at pH 6.0, followed by 4% formaldehyde at pH 10.5;
both solutions containing pick acid); 2, etching of the sections prior to the immunoincubation
(1-2
seconds in a saturated solution of NaOH in ethanol); 3, pretreatment of the sections with Triton X100 (0.1 %); 4, reduction of the NaCl content of the vehicle buffer to 30 n&I. Confirming a series
of previous reports, the use of secondary Fab fragments (rather than IgGs) and small gold particles
(5 - 10 nm, or lnm followed by silver enhancement) gave superior results in terms of sensitivity.
Digital images were obtained with an image analysis program (analySIS; Soft Imaging Systems
Gmbh, Mtinster, Germany) that had been modified for the acquisition of high-resolution digital
images [3] and evaluation of membrane-associated
immunogold labelling [2].
In the brain and retina the immunogold labelling was concentrated along those glial cell
membrane domains that were apposed to blood vessels (Fig. 1A) or to pia (glia limitans, GL, Fig.
1B). Weak labelling occurred in endothelial cells (End, Fig. 1A) where the gold particles appeared
to be associated preferentially
with caveola-like
structures. A quantitative analysis of the
immunolabelling
of the Mtiller cell (the main type of glial cell in the retina) indicated a highly
differentiated compartmentation
of the AQP4 water channel with a -10 fold enrichment in
perivascular and vitreal endfeet (Fig. 1C). Our findings suggest that the latter membrane domains
may play a crucial role in the handling of water in the central nervous system [2].
1. Van Lookeren Campagne M. et al., J. Histochem. Cytochem. 39 (1991) 1267-1279.
2. Nagelhus E.A. et al.. J. Neurosci. 18 (1998) 2506-25 19.
3. Haug F.-MS. et al., Proceedings: microscopy and microanalysis (edited by G.W. Bailey et al.), pp.
618-619. San Francisco Press (1996).
Acknowledgement: This work was done in collaboration with Dr. Soren Nielsen, University of Aarhus,
Denmark, and Dr. Peter Agre, Johns Hopkins University School of Medicine, Baltimore, U.S.A.
Vitreal endfeet
membr. facing basal lam.
GCL
IPL
lateral membr.
Perivascular endfeet
INL
Perivascular endfeet
OPL
ONL
Photoreceptor IS
PhL
0
C
5
10
15
20
Gold particles/pm
1
25