(E) Double immunostaining technique
In addition to its very high sensitivity, the great advantage of our technique is to afford the opportunity of determining by a second immunohistochemical procedure the neurotransmitter content of the identified pathway. For this purpose, we have developed a double immunostaining procedure on the same sections including the combination of colchicine treatment and retrograde tracing by CTb.
We have shown above that the retrograde labeling of CTb is complete 24 h after injection. Therefore, we administered colchicine at this time by means of two previously implanted cannulae. We tested the effect of 24 and 48 h of colchicine treatment on the retrograde tracing (see above) and on the immunohistochemical identification of neurotransmitters and their synthesizing enzymes. We determined that the colchicine treatment only slightly increased the staining intensity of the neurons when using antisera to the enzymes ChAT, TH and PNMT. In contrast, the number of immunostained cells as well as their staining intensity dramatically increased for the neuropeptides and the neurotransmitters like serotonin, the more so with a 48 h colchicine treatment.
The identification of the histochemical nature of the retrogradely labeled cells is based on the differential aspect of CTb-(punctate) and neurotransmitter (homogeneous)-immunoreactive material in the cell bodies that are well contrasted by the use of two different chromogens (DAB-nickel and DAB) on the same sections for the first and second immunostaining respectively. The double-labeled cells were then easily recognized by the presence of black punctate granules over a brown diffusely stained cytoplasm (Fig. 5).
The straightforward identification of the double-labeled cells requires at least 3 imperative conditions:
(1) the second immunohistochemical procedure should not cross-react with the first, thereby avoiding the observation of false double-labeled neurons;
(2) the black punctate reaction products of CTb should not completely fill the cell bodies whereby observation of the brown deposit becomes difficult;
(3) the homogeneous brown deposit of the second immunohistochemical procedure should not mask the black CTb punctate reaction product.
To eliminate the first possibility, we used different species antisera directed against CTb (goat) and the neuroactive substances (rabbit). We selected second antisera that had been passed through agarose gels containing immobilized serum proteins from the other species to maximally minimize cross-reactivity between the two procedures. We also used the highly sensitive streptavidin-HRP reagent to detect CTb and the classical PAP technique for the neurotransmitters and their synthesizing enzymes. In this way, we completely eliminated the cross-reactivity between the two immunohistochemical procedures. Indeed, we observed an unacceptable artefactual labeling and cross-reaction when using the streptavidin-HRP for the two procedures while we obtained only a faint retrograde labeling of CTb when using the goat PAP (see above). In contrast, the use of rabbit PAP for the immunohistochemical detection of the neuroactive substances, although less sensitive than streptavidin-HRP, gave rise to an extensive and intense labeling of the cell bodies.
With regard to the last two points, first, we selected the appropriate procedure to detect CTb, i.e. with the maximal sensitivity but giving a granular staining of theretrogradely labeled cells, without any background (see above). Secondly, we determined the appropriate dilution for each antibody directed against the neuroactive substances (see Materials and Methods) as well as for the IgG (1:400-800) and the rabbit PAP (1:400-1000) in order to obtain a homogeneous brown deposit sufficiently light to avoid masking of the black granules of CTb. The color photomicrographs shown in Fig. 5 illustrate representative examples of double-labeled neurons containing black granules of CTb material and a brown homogeneous deposit. Note that the double labeled cells can be easily identified even at a low magnification (Fig. 5A) and that the neurons containing only CTb punctate retrograde labeling did not show any trace of brown deposit, thus indicating the absence of cross-reaction between the two successive immunohistochemical procedures.