Neuroscience Letters, 85 (1988) 169 171
169
Elsevier Scientific Publishers Ireland Ltd.
NSL 05130
The glucose oxidase-DAB-nickel method in
peroxidase histochemistry of the nervous system
Siyun Shu, G o n g Ju and Lingzhi F a n
Department ~[ Neurohiology, The Fourth Military Medical University, Xi'an (The People's Republic ~[
China)
(Received 6 October 1987: Accepted 24 October 1987)
Key words: Immunohistochemistry: Horseradish peroxidase (HRP): Diaminobenzidine IDAB): Glucose
oxidase: Nickel
A combination of the glucose oxidase diaminobenzidine (DAB) method and the DAB nickel method
can successfully bring out details of immunoreacti,m structures in immunostained preparations. It is especially beneficial for visualizing fibers and terminals.
Diaminobenzidine (DAB) was first used as a chromogen for peroxidase histochemistry by Graham and Karnovsky [2] and was later widely used in neuronal tract
tracing and immunohistochemistry. Hancock [3] has developed the DAB nickel
method for immunohistochemistry; with the addition of nickel ammonium sulfate
the reaction products turned black and the details of immunostained structures were
better displayed. For peroxidase histochemistry, hydrogen peroxide is usually added
directly to the incubation medium. Itoh et al. [5], while describing a method for electron microscopic demonstration of horseradish peroxidase (HRP), recommended the
use of glucose oxidase and glucose instead of H20:. We have applied glucose oxidase
to the DAB--nickel method in immunohistochemistry and found it very successful
in bringing out nerve fibers and terminals.
Young adult Sprague-Dawley rats were used. All surgical procedures were performed under general anesthesia with sodium pentobarbital (35 mg/kg b.wt., i.p.).
The animals received a dose of about 150/tg of colchine intraventricularly 2 days
prior to perfusion-fixation with 476 paraformaldehyde and 0.1% picric acid. The
brains were frozen and sectioned at 40/lm. The sections were incubated in l:1000
rabbit antiserum against vasoactive intestinal peptide (VIP, Penninsula) for 3 days
and then processed with peroxidase antiperoxidase (PAP) [10] or avidin biotin complex (ABC) (Vector) technique [4]. Subsequently, the sections were further treated
with 0.05 % DAB [9] or the glucose oxidase-DAB--nickel (GDN) method.
Correspondence: G. Ju, Department of Neurobiology, The Fourth Military Medical University, Xi'an.
Shaanxi, The People's Republic of China.
0304-3940/88,'$ 03.50 @ 1988 Elsevier Scientific Publishers Ireland Ltd.
170
Procedure of the GDN method. After incubated with the primary antibody and
treated with the PAP or ABC method, the sections were processed as follows:
(1) Collect sections in phosphate buffer saline.
(2) Rinse in 0.1 M acetate buffer (pH 6.0).
(3) Incubate in G D N solution for about 20 minutes.
A: Nickel ammonium sulfate
2.5 g
0.2 M acetate buffer (pH 6.0)
50 ml
B: 3,3'-diaminobenzidine dihydrochloride
50-70 mg
H20
50 ml
Mix A and B solutions before use and add:
/C-o-Glucose
200 mg
Ammonium chloride
40 mg
Glucose oxidase (Sigma, type VII)
0.5 1 mg
(4) Rinse in acetate buffer and mount.
Both DAB and G D N methods showed dense VIP-immunoreactive terminals in the
bed nucleus of the stria terminalis (Fig. 1A,B), the terminals, however, were stained
Fig. 1. VIP immunoreactivityof the bed nucleus of the stria terminalis. A: DAB method. B: GDN method.
Bar = 50/Lrn.
Fig. 2. VIP immunoreactivityof the frontal cortex. Bar = 50 am.
Fig. 3. VIP immunoreactivityof the frontal cortex, high magnification, showing terminals and dendritic
spines. Bar = 10/tm.
171
much d e n s e r with the G D N m e t h o d . In less i m m u n o r e a c t i v e a r e a s the difference
between these 2 m e t h o d s was even m o r e striking. Figs. 2 a n d 3 illustrate the staining
o f the f r o n t a l cortex. T h e y clearly s h o w the a d v a n t a g e s o f the G D N m e t h o d , the
nerve processes, the t e r m i n a l s a n d even the d e n d r i t i c spines being beautifully displayed.
D A B was first i n t r o d u c e d for n e u r o n a l tract tracing technique by K r i s t e n s s o n et
al. [6] a n d LaVail a n d L a V a i l [7]. L a t e r several o t h e r c h r o m o g e n s a p p e a r e d in the
literature, claimed to be m o r e sensitive t h a n D A B , such as o-dianisidine, benzidine.
H a n k e r Y a t e s reagents a n d t e t r a m e t h y l b e n z i d i n e [9]. D u e to its n o n - c a r c i n o g e n i c i t y
and great sensitivity, t e t r a m e t h y l b e n z i d i n e has largely replaced D A B for light microscopic tract tracing studies. D A B , however, r e m a i n s one o f the reagents o f choice
in m a n y electron m i c r o s c o p i c ( E M ) studies a n d especially in i m m u n o h i s t o c h e m i c a l
studies. T h e r e are several w a y s to intensify D A B reaction. A d a m s [1] r e c o m m e n d e d
the use o f cobalt. H a n c o c k [3] d e v e l o p e d the nickel m e t h o d to p r o v i d e better i m m u n o s t a i n i n g o f nerves fibers a n d terminals. In the D A B m e t h o d , h y d r o g e n p e r o x i d e
is usually a d d e d directly to the i n c u b a t i o n m e d i u m . A high c o n c e n t r a t i o n o f h y d r o gen p e r o x i d e , however, m a y be deleterious to the e n z y m e p e r o x i d a s e a n d tissue. Itoh
et al. [5] r e c o m m e n d e d the use o f glucose oxidase a n d glucose instead to a v o i d high
c o n c e n t r a t i o n o f h y d r o g e n peroxide. T h e use o f glucose oxidase a n d glucose was first
exploited by L u n d q u i s t a n d Josefsson [8] as a sensitive m e t h o d o f d e t e r m i n i n g peroxidase activity in tissue a n d was c l a i m e d to increase the sensitivity o f the reaction. The
rationale b e h i n d it, as was suggested by I t o h et al. [5], m a y lie in the fact that d u r i n g
glucose o x i d a t i o n reaction, there occurs a c o n t i n u o u s release o f h y d r o g e n peroxide,
thus favors the d e p o s i t i o n o f D A B reaction p r o d u c t s a r o u n d H R P molecules. The
a p p l i c a t i o n o f glucose o x i d a s e to the D A B - n i c k e l m e t h o d , as is p r o p o s e d in the present study, has been p r o v e d to be successful in b r i n g i n g out details o f i m m u n o r e a c tive structures. It is especially beneficial for visualizing nerve fibers a n d terminals.
I Adams, J.C., Technical considerations on the use of horseradish peroxidase as a neuronal marker,
Neuroscience, 2 (1977) 141 145.
2 Graham, R.C., Jr. and Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J.
Histochem. Cytochem., 14 (1966) 291 302.
3 Hancock, M.B., Visualization of peptide-immunoreactive processes of serotonin-immunoreactive cells
using two color immunoperoxidase staining, J. Histochem. Cytochem., 32 (1984) 311 314.
4 Hsu, S.-M., Raind, L. and Fanger, H., Use of avidin-biotin-peroxidasecomplex (ABC) in immunoperoxidase technique, J. Histochem, Cytochem., 29 (1981) 577 580.
5 Itoh, K., Konishi, A., Nomura, S., Mizuno, N, Nakamura, Y. and Sugimoto, T., Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method, Brain Res., 175 (1979) 341 346.
6 Kristensson, K., Olsson, Y. and Sj6strand, J., Axonal uptake and retrograde transport of exogenous
proteins in the hypoglossal nerve, Brain Res., 32 (1971) 399~406.
7 LaVail, J.H. and LaVail, M.M., Retrograde axonal transport in the central nervous system, Science,
176(1972) 1416 [417.
8 Lundquist, I. and Josefsson, J.-O., Sensitive method for determination of peroxidase activity in tissue
by means of coupled oxidation reaction, Anal. Biochem., 4l (1971) 567 577.
9 Mesulam, M.-M., Tracing Neural Connections with Horseradish Peroxidase, Wiley, New York, 1982,
pp. 1 151.
10 Sternberger, L.A., tmmunocytochemistry, Prentice Hall, Englewoods Cliffs, N J, 1974.