A . Velasco et al. Brain Research 883 2000 98 –106
99
detected in the goldfish optic nerve with molecular weights with 3 H O in 100 methanol for 10 min. After rinsing
2 2
of 145 kDa [16,30]. However, NF-H proteins have not twice for 10 min in phosphate buffer saline solution with
been identified in the optic nerve of goldfish [13]. These 0.2 Triton X-100 PBS–Tx, the sections were incubated
differences with regard to the mammalian proteins appear overnight with RT97 monoclonal antibody Hybridoma
to be important in the continued plasticity of the visual Bank Iowa University at a dilution of 1:1000 at 188C in a
system [13]. Several of these proteins, such as plasticin humidity chamber, and subsequently incubated at room
and gefiltin, modify their expression during regeneration temperature with biotinylated mouse IgG Vector at a
[12,14,15,27,28,33]. Nevertheless, in the visual system of dilution of 1:200 and avidin–peroxidase complex Vector
mammals [5,10,36] and several anamniotes, such as 1:250. The sections were finally reacted with 0.025
Xenopus and trout [6,38], proteins of NF-H in the axon 3.39-diaminobenzidine
tetrahydrochloride DAB
and ganglion cells have been detected. These proteins are also
0.025 hydrogen peroxide in 0.2 M Tris–HCl pH 7.6 found in other areas of fish brains [1,9,22].
for 10 min at room temperature. Sections were dehydrated The antibody RT97 has been used to recognize the
and mounted in Entellan Merck. The right retinas and phosphorylated 200-kDa neurofilament sub-unit in mam-
optic nerves of lesioned tench and the visual pathways mals [37]. Furthermore it also recognizes, though with
retinas, optic nerves and optic tecta of unlesioned tench lesser affinity, another phosphorylated neurofilament pro-
were used as controls. Immunohistochemical controls were tein of 155 kDa and also tau of Alzheimer neurofibrillary
performed either by the omission of the primary antibody tangles [2,3,8,21] and the upper band of the MAP1B
or by replacement with a non-immune serum. doublet [19]. This antibody has also been used to label
specific populations of neurons in the rat [20,23]. In this 2.1. Immunoprecipitation Western blot
study, we analyze the distribution and modification of the immunostaining of RT97 and which proteins are recog-
2.1.1. Tissue preparation nized by this antibody in the normal and damaged tench
Retina, optic nerve and optic tectum from control and visual system.
lesioned tench 30 days post-crushing were dissected and isolated. Tissue was sonicated twice at 48C for 5 s in lysis
buffer which contained 50 mM Tris HCl, pH 7.4, 150 mM
2. Material and methods
NaCl, 1 Triton X-100, 1 mM EGTA, 0.4 mM EDTA, 2.5 mg ml aprotinin, 2.5 mg ml leupeptin, 1 mM PMSF and
We used 60 tench Tinca tinca L., Teleostei, of body 0.2 mM Na VO . Tissue homogenates were centrifuged at
3 4
length 15–20 cm, which were obtained from a commercial 15 000 g at 48C for 20 min. The soluble protein con-
hatchery. The animals were anesthetized with 0.03 centration was determined using BioRad Reagent.
tricaine methanesulphonate MS222, Sigma. The left optic nerve was crushed 1 mm from the eyeball with a fine
2.1.2. Immunoprecipitation watchmaker’s forceps for about 3 s. The animals were kept
A total of 350 mg of protein in each sample of tissue in aquaria at 18–208C for periods from 1 to 200 days. At
were incubated over-night at 48C with 12 ml of anti-RT97 least five animals were used at each different time point 2,
antibody or 12 ml of anti-MAP1B monoclonal antibody 7, 15, 30, 45, 60, 90, 120, 150 and 200 days after crushing
Clone AA6, RBI, USA. Immunocomplexes were precipi- the nerve. Animal manipulations were performed in ac-
tated with 25 ml of protein A conjugated to agarose cordance with the directives of the European Communities
Upstate Biotechnology Inc., USA. When anti-MAP1B Council 86 609 EEC and current Spanish legislation
was used to immunoprecipitate, the same amount of a BOE 67 8509-12, 1988 for animal care and ex-
rabbit anti-mouse IgG Pierce was added to increase the perimentation.
affinity of the immunocomplex for protein A-agarose. The The control and lesioned animals were re-anesthetized
immunoprecipitates were washed three times at 48C with and perfused transcardially with 0.68 NaCl solution
the lysis buffer containing proteases and phosphatase followed by 4 paraformaldehyde in phosphate buffer 0.1
inhibitors. Laemmli buffer was added to the immuno- M, pH 7.4 PB. The retina, optic nerve and optic tectum
precipitates and the mixture was denatured by boiling. were dissected out and post-fixed for 4 h at 48C in the
same fixative solution. The sampled tissues were then rinsed in PB and soaked in 30 sucrose in PB overnight.
2.1.3. Electrophoresis, electrotransfer and Western blot The tissues were then frozen in liquid nitrogen and cut on
Denatured proteins were subjected to 7.5 sodium a cryostat in 15–20 mm-thick sections. The brains were
dodecyl sulfate–polyacrylamide gel electrophoresis SDS– sectioned perpendicularly to the longitudinal plane, while
PAGE and then electrophoretically transferred to nitro- the optic nerves were sectioned longitudinally and trans-
cellulose membrane. Immunoblotting was carried out as versely. The sections were then mounted on gelatin-coated
described previously [7] with 1:1000 dilution of anti-RT97 slides and washed in PB before immunohistochemical
antibody and horseradish peroxidase-coupled secondary processing. Endogenous peroxidase activity was blocked
antibody Pierce, USA. Labeled proteins were detected
100 A
with Super Signal West Pico chemiluminescent substrate proteins as shown in Fig. 1A, lane 2, there appears to be an
Pierce, USA. induction of the labeling of a band of approximately 325
kDa. There was no appreciable change in the labeling of 2.2. Digitally processed images
the 335 kDa protein. Images of processed tissues were obtained with an
3.1.2. Optic nerve Olympus DP10 digital camera coupled to a Leica DMRB
Proteins from nerve lysates were immunoprecipitated photomicroscope. Immunoblot-films were directly di-
with anti-RT97 antibody followed by Western blot with the gitally-scanned. Original pictures were further processed
same antibody. As shown in Fig. 1B, the pattern of optic with Adobe Photoshop
E 5.5 software so as to obtain the nerve proteins labeled in tench is dramatically altered after
optimal contrast within the same figure plate. crushing. In control tench there is a modest labeling of a
115 kDa protein lane 1. Optic nerve crushing resulted in a clear and marked increase in the labeling of this protein
3. Results Fig. 1B, lane 2. Interestingly, there appears to be a
