Histochemical Society, Inc
pp. 1079-1084. 1995
Simple Technique for Staining
Imidazole and Osmium Tetroxide'
GEORGES THIERY, JACQUES BERNIER, and MICHEL BERGERON2
Universiii de MoniriaL, Montriad Qu6&ec, Canada.
Received for publication February 8, 1995 and in revised form May 3, 1995; accepted May 25, 1995 (5T3592).
We describe a simple new technique based on the affinity
of imidazole and osmium tetroxide for unsaturated lipids.
Organs (e.g., kidney, liver, intestine) were perfused in vivo
with a glutaraldehyde solution. Tissue fragments were then
immersed in a solution containing imidazole and
are further stained with
double lead and copper citrate
pm) or thick (0.1-0.3 pm) sec-
tions were observed with transmission electron microscopy
(80-100 kV). The method presented permits excellent visu-
alization of cell membranes (e.g., endoplasmic reticulum,
of en bloc staining coupled with thick sections (0.2-0.5
pm) has provided new views on the spatial organization of cell
ganelles in the cytoplasm, i.e., mitochondria
and endoplasmic reticulum
However, metal impreg-
nation has a major drawback because it cannot offer clear images
of cell membranes and
the relationships among organelles
cannot be observed with precision. When post-fixed with potas-
sium ferrocyanide and osmium
cell membranes could be visual-
sections. However, because the many plasma
membrane infoldings act as a barrier to resin penetration, the sec-
tions become too fragile to resist the electron beam and are diffi-
cult to observe. We present a new method, based on the affinity
of imidazole and
for unsaturated lipids, that has been al-
ready demonstrated by Angermuller and Fahimi (9). This method
appears to achieve better resin penetration than osmium/ferro-
cyanide and therefore produces excellent contrast for membrane
Supported by grant MT-2862 from the Medical Research Council of
Canada and by the Minisdie de I'Education of the Government of Qut-
Correspondence to: Dr. Michel Bergeron, Dept. de physiologie,
Facultt de Medecine. Universite de Montrtal, CP 6128, Succ. Centre-ville,
Montreal, Quebec, Canada, H3C 3J7.
endocytotic apparatus) because it favors good resin penetra-
tion and the alkaline pH preserves cell volume. A better
stereomicroscopic analysis of the relationship between cell
carried out with thick sections. The im-
cal steps are easy and simple to follow. Furthermore, it
complement other cytochemical methods.
Endoplasmic reticulum; Cytomembranes; Histochemis-
try; Kidney; Liver; Rat; Imidazole; Osmium tetroxide.