Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons
Luppi P.H., Fort P., Jouvet M.
Brain Res. 534 (1-2) pages : 209-224 (1990)


Materials and Methods

Materials and Methods


(A) Injection sites

(B) Retrograde labeling

(C) Artefactual labeling due to uptake by fibers of passage

(D) Anterograde tracing

(E) Double immunostaining technique



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Figure 2A

A: overview of a frontal section at the level of the medulla oblongata showing a typical iontophoretic injection site in the nucleus reticularis parvicellularis (case K120) using a 2-µA pulsed positive current for 30 min.
Bar= 2 mm.

B: higher magnification of the site shown in A precisely illustrating its extent and localization. The stars and arrows designate the same vessels or fiber bundle in B and D.
Bar = 400 µm.

D: adjacent section to that shown in B at a higher magnification, counterstained with Cresyl violet and immunostained with serotonin, showing the presence of counterstained cell bodies and serotonin immunoreactive fibers in the site proving the absence of tissue necrosis.
Bar =50 µm.

C: photomicrograph illustrating retrogradely labeled cells in the medial (left part) and lateral (right part) divisions of the nucleus of the solitary tract. Note also the presence of punctate anterograde labeling in the same areas.
Bar = 100 µm.

E: photomicrograph showing anterogradely labeled fibers organized as a bundle running dorsally to the facial nucleus approximately 2 mm rostral to the site shown in A and B. Varicose fibers inside the facial nucleus sometimes formed basket-like structures around motoneuron somata (arrow).
Bar = 200 µm.

F: photomicrograph showing the great number of anterogradely labeled varicose fibers in the hypoglossal nucleus after the CTb iontophoretic injection shown in A and B.
Bar = 100 µm.

To optimize the CTb immunostaining shown in C, E and F, the sections were incubated 3-4 days in CTb antiserum (1: 40,000) at 4 °C followed by sequential incubations overnight at 4 °C in the biotinylated donkey anti-goat IgG (1:2,000) and the streptavidin-HRP (1:40,000). Note that with such maximal conditions, we obtained a weak counterstaining of the sections ideally suited for accurate localization of CTb immunostained material.

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