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
selective labeling of several other proteins in the optic 3.1. Immunoblotting Western blot
nerve induced after the lesion 105, 160, 200, 325 and 335 kDa, approximately.
3.1.1. Retina Proteins from retina lysates were immunoprecipitated
3.1.3. Optic tectum with anti-RT97 antibody followed by Western blot with the
As seen in Fig. 1C lane 1, the anti-RT97 recognized at same antibody. As shown in Fig. 1A, lane 1, the anti-
least three proteins of different molecular weights in RT97, in normal conditions, clearly recognized at least
control animals 105, 115 and 200 kDa approximately. three proteins of different molecular weights: 115, 200 and
The optic nerve crushing Fig. 1C, lane 2 did not modify 335 kDa approximately. The retina protein labeling pattern
the labeling of the 105 and 115 kDa proteins appreciably, in tench was altered by optic nerve crushing. While there
but induced a modest and selective labeling of a single was a decrease in the labeling of the 115 and 200 kDa
high molecular weight protein 325 kDa, approximately.
Fig. 1. A Western blot WB with RT97 antibody a-RT97 after immunoprecipitation IP with a-RT97 of 350 mg of proteins of retina lysates of normal lane 1 and lesioned lane 2 tench at 30 days after optic nerve crushing. Three proteins were labeled with RT97 antibody in normal tench retina with
molecular weights of 115, 200 and 335 kDa approximately lane 1. In lesioned tench lane 2, the intensity of the a-RT97 labeling decreased in two bands arrows and a new stained band of approximately 325 kDa appeared arrow with asterisk. B Western blot with a-RT97 after immunoprecipitation with
a-RT97 of 350 mg of proteins from optic nerve lysates from normal lane 1 and lesioned lane 2 tench 30 days after crushing. In the normal optic nerve, only a 115 kDa molecular weight protein was labeled lane 1. At 30 days after the lesion lane 2, the immunoreactivity of the 115 kDa band was
increased notably and other proteins of 105, 160, 200, 325 and 335 kDa, approximately arrows were labeled. C Western blot with RT97 antibody after immunoprecipitation with a-RT97 lanes 1 and 2 and MAP 1B lane 3 antibodies of 350 mg of proteins from optic tectum lysates of normal lane 1 and
lesioned tench lanes 2 and 3 30 days after crushing. At least three different proteins were recognized with RT97 in normal animals lane 1. After crushing lane 2, the immunoreactivity of the 200 kDa protein disappeared arrow and the labeling of a protein of approximately 325 kDa was induced
arrow with asterisk. Proteins immunoprecipitated with MAP1B antibody that were immunoreactive with RT97 antibody by Western blot in optic tectum from lesioned tench are shown in lane 3.
A . Velasco et al. Brain Research 883 2000 98 –106
101
The slight labeling of the band at approximately 200 kDa were seen to run along the more vitreal portion of the
in control conditions disappeared after crushing the optic retina from the peripheral zone towards the optic nerve
nerve. head penetrating the central zone close to the central artery
Fig. 2B. 3.1.4. Anti-MAP1B antibody
In the control optic nerve, RT97-immunolabeling was In order to study which proteins recognized by the RT97
weak and diffuse throughout the nerve, with the exception antibody are also recognized by the MAP1B antibody in
of a small group of axons which were strongly labeled in the tench visual system, we performed an additional
one of the nerve folds towards the interior part of the nerve experiment. We carried out an immunoprecipitation of
Fig. 3A. proteins from the retina, nerve and optic tectum with the
In the optic tectum OT RT97 labeled axons were MAP1B or RT97 antibodies followed by Western blot with
located in the stratum marginale SM, stratum opticum RT97 antibody. Only one representative result from tench
SO, the stratum fibrosum and griseum superficiale tissue, optic tectum, is shown in Fig. 1C, lane 3. This
SFGS and stratum griseum centrale SGC Fig. 5A. In experiment demonstrated that the RT97 antibody, in our
the deeper zone of the SO and in the SFGS the immuno- conditions, could also recognize proteins purified with the
reactive axons were organized in laminas whereas in the MAP1B antibody in tench visual system.
SGC the axons were isolated Fig. 5A. 3.2. Immunohistochemistry
3.2.2. Lesioned animals After optic nerve crush, we did not find modifications in
3.2.1. Control animals the labeling with RT97 in the retina.
In control retinas, the RT97 antibody labeled horizontal In the optic nerve,
1 day after lesion the diffuse labeling cells and the growing ganglion cell axons that were being
of the nerve disappeared in the area of the lesion, but was formed in the peripheral portion intensely Fig. 2. The
maintained in the rest of the nerve, both in the portion horizontal cells, located in the more vitreal level of the
proximal to the eye and in the portion distal to the eye outer nuclear layer ONL, showed immunoreactivity in
toward the optic tract Fig. 3B. In the first week after their perikarya and processes, which also penetrated the
lesion diffuse staining was present in the area between the inner nuclear layer Fig. 2A. The labeled growing axons
retina and the zone of lesion, but disappeared completely
Fig. 2. RT97 labeling in the retina A and optic nerve head B of control tench. A RT97 positive horizontal cells in the inner nuclear layer INL. Somata and several processes arrows were stained. Scale bar: 25 mm. B Intensely positive ganglion cell axons in the vitreal zone of the retina
penetrating the optic nerve head arrows surrounding the central artery V. Scale bar: 250 mm.
102 A
Fig. 3. Longitudinal A, B, C, D, G and transverse E, F sections through the optic nerve showing the variation of the labeling of RT97 in the normal tench and at different times after crushing the left optic nerve. c indicates the site of the crush, p is proximal, and d distal to the eyeball. Scale bar: 250
mm. A Normal optic nerve. Strongly labeled axons arrows correspond to the growing axons of the retina. B One day after crushing the labeling disappeared in the zone of lesion, but remained in the proximal and distal zones. C Fifteen days after crushing, intensely labeled fibers are observed near
the crush site arrow. D Strongly labeled axons cross the lesion zone at 30 days post-crush. E, F Transverse sections of lesioned optic nerve 45 days after the lesion. E Weak labeling of the fibers in the area proximal to the retina. F Strongly positive axons in the distal zone of the nerve adjacent to the
lesion region. G Intensely positive fibers at 90 days post-crush run through the optic nerve.
A . Velasco et al. Brain Research 883 2000 98 –106
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both in the lesion zone and in the rest of the nerve towards distribution of the labeling remained until 200 days post-
the optic tract. At 15 days, in the proximal area of the
lesion. optic nerve the diffuse labeling was more intense while
strongly labeled isolated axons were observed running from the retina towards the crush zone Fig. 3C. Several
of these immunopositive axons crossed the damaged area,
4. Discussion
