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Anti CTL1 antibody could detect the full-length (200-k Da) recombinant L1 in the lysate of transfected High5 insect cells (lane 3), whereas no positive band was detectable in the mock-transfected (control) High5 lysate (lane 4). Omission of primary antibody (-anti CTL1) resulted in no immunostaining (substantia nigra). Blocking of anti CTL1 antibody with epidermal growth factor (EGF) did not interfere with the immunostaining in the neighboring section of (b). Absorption of the antibody with L1 gene-transfected membrane (L1m) completely eliminated the immunostaining, whereas absorption of the antibody with mock-transfected membrane (Mock) had no effect (d'-f').
d,d': stria terminalis; e,e': substantia nigra; f,f': cerebellar cortex. The two polyclonal antisera (anti CTL1 and anti FLL1 antibodies) demonstrated an analogous staining pattern on immunohistochemistry, though the latter antiserum tended to stain passing proximal axons better than the former (Fig. Consequently, described here are the results obtained with the anti CTL1 antibody unless specified otherwise.
e and f, Bright-field photomicrograph showing L1 neurons in the lateral posterior thalamic nucleus of the upper brainstem.
a,a',b,b': Bar = 25 μm; c,c',d,d',e: Bar = 200 μm; f: Bar = 50 μm. The immunoreactivity of fibres was generally strong and this type of staining was observed chiefly in the major fibre bundles.
To examine the fine structure more precisely, selected areas with this type of staining were chosen and subjected to an immuno-electron microscopic procedure.SP control; control side of the same section immunostained with anti SP antibody.c,c' and d,d' No change was found on L1 in the distal side of the lesion of the bundle (c'; L1 knife cut), though substance P immunoreactivity was clearly diminished (d'; SP knife cut, open arrowheads).Here, we identified novel sites of L1 immunoreactivity in various regions of the brain., lane 4).
A clear single 200 k Da band was seen in L1-transfected cell lysate, but not mock-transfected cell lysate using the anti CTL1 antibody.The aim of this experiment is to observe whether L1 immunoreactive protein accumulates at the transected axons.