Summary
p-Phenylenediamine/pyrocatechol mixture (PPD-PC) was evaluated as a reagent for the ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase (HRP). HRP crystals were applied to the proximal stumps of the severed infraorbital nerves in rats. After 48 h the rats were sacrificed by perfusion, and the trigeminal ganglia ipsilateral to the severed nerves were processed for HRP cytochemistry and then prepared for electron microscopy. PPD-PC was rapidly oxidized in HRP-labeled neurons to form a dark brown-black osmiophilic reaction product which was more readily visible than the DAB product in the sections. This facilitated selection by light microscopy of areas in the epoxy wafers for ultrathin sectioning. In thin sections viewed under the electron microscope, the osmicated electron opaque PPD-PC reaction product was present in membrane-bound structures including smooth endoplasmic reticulum and granules of various sizes. The PPD-PC reaction product formed after 10-min incubation appeared to be more electron opaque than the DAB reaction product formed after 20 min. PPD-PC was found to be much less readily oxidized than DAB by endogenous hemoproteins. This methodology facilitated the ultracytochemical localization of HRP in neurons following retrograde axonal transport.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bussolati G (1971) Histochemical demonstration of sulfate groups by means of diaminobenzidine. Histochem J 3:445–449
Coleman RA, Ramp W, Toverud S, Hanker JS (1976) Electron microscopic localization of lactate dehydrogenase in osteoclasts of chick embryo tibia. Histochem J 8:543–558
Droz B (1975) Synthetic machinery and axoplasmic transport: Maintenance of neuronal connectivity. In: Brady RO (ed) The basic neurosciences. Raven Press, New York. (Tower DB (ser ed) The nervous system, vol 1, pp 111–127
Graham RC Jr, Karnovsky MJ (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of the mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291–302
Gregg JM, Dixon AD (1973) Somatotopic organization of the trigeminal ganglion in the rat. Arch Oral Biol 18:487–498
Griswold DP Jr, Casey AE, Weisburger EK, Weisburger JH (1968) The carcinogenicity of multip intragastric doses of aromatic and heterocyclic nitro or amino derivatives in young female Sprague-Dawley rats. Cancer Res 28:924–933
Hanker JS (1979) Osmiophilic reagents in electron microscopic histocytochemistry. Prog Histochem Cytochem 12:1–85
Hanker JS, Yates PE, Metz CB, Rustioni A (1977) A new, specific, sensitive, and noncarcinogenic reagent for the demonstration of horseradish peroxidase. Histochem J 9:789–792
Hayat MA (in press 1981) Fixation for electron microscopy. Academic Press, New York
Jones EG, Hartman BK (1978) Recent advances in neuroanatomical methodology. Annu Rev Neurosci 1:215–296
Kitai ST, Kocsis JD, Preston RJ, Suginori M (1976) Monosynaptic inputs to caudate neurons identified by intracellular injection of horseradish peroxidase. Brain Res 109:601–606
Kristenson K, Olsson Y (1976) Retrograde transport of horseradish peroxidase in transected axons: 3. Entry into injured axons and subsequent localization in perikarya. Brain Res 115:201–213
LaVail JH, LaVail MM (1974) The retrograde intra-axonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study. J Comp Neurol 157:303–357
Nandy K (1971) Properties of neuronal lipofuscin pigment in mice. Acta Neuropathol 19:25–32
Nauta HJW, Kaiserman-Abramoff IR, Lasek RJ (1975) Electron microscopic observations of horseradish peroxidase transported from the caudoputamen to the substantia nigra in the rat: possible involvement of the agranular reticulum. Brain Res 85:373–384
Peters A, Palay SL, Webster H de F (1976) The fine structure of the nervous system. WB Saunders, Philadelphia, p 37
Richardson K, Jarret L, Finke E (1960) Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol 35:313–323
Roels F, Goldfischer S (1971) Staining of nucleic acids with 3,3′-diaminobenzidine (DAB). J Histochem Cytochem 19:713–714
Romanovicz DK, Hanker JS (1977) Wafer embedding: specimen selection in electron microscopic cytochemistry with osmiophilic polymers. Histochem J 9:317–327
Samorajski T, Keefe JR, Ordy JM (1964) Intracellular localization of lipofuscin age pigments in the nervous system. J Gerontol 19:262–276
Sato T (1968) A modified method for lead staining of thin sections. J Electron Microsc 17:158–159
Somogyi P, Hodgson AJ, Smith AD (1979) An approach to tracing neuron networks in the cerebral cortex and basal ganglia. Combination of Golgi staining, retrograde transport of horseradish peroxidase and anterograde degeneration of synaptic boutons in the same material. Neuroscience 4:1805–1852
Sotelo C, Richie D (1974) The smooth endoplasmic reticulum and fast orthograde transport of horseradish peroxidase in the nigro-striatalnigral loop. Anat Embryol (Berl) 146:209–218
Straus W (1964) Factors affecting the cytochemical reaction of peroxidase with benzidine and the stability of the blue reaction product. J Histochem Cytochem 12:462–469
Winer JA (1977) A review of the status of the horseradish peroxidase method in neuroanatomy. Biobehav Rev 1:45–54
Author information
Authors and Affiliations
Additional information
Supported by NIH Grants DE 04730, DE 02668 and DE 00288 from the National Institute for Dental Research, NIH Grant RR 0533 from the Division of Research Facilities and Resources, and a grant to the Neurobiology Program from the Alfred P. Sloan Foundation
Rights and permissions
About this article
Cite this article
Carson, K.A., Lucas, W.J., Gregg, J.M. et al. Facilitated ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase in peripheral nerve. Histochemistry 67, 113–124 (1980). https://doi.org/10.1007/BF00493230
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00493230