Free Horseradish Peroxidase Research Articles

A re-examination of the rubro-olivary tract in the cat, using horseradish peroxidase as a retrograde and an anterograde neuronal tracer

Most authors using free horseradish peroxidase as a retrograde tracer have had difficulties in demonstrating a rubro-olivary tract. Injections of this tracer into the inferior olive in animals of different species resulted in only a few retrogradely labelled rubral cells within the rostral part of the ipsilateral nucleus. The present study, which is based on the highly sensitive technique of Mesulam (1978), confirms this observation. Injections of free horseradish peroxidase (Serva or Sigma VI type) into the dorsal lamella of the principal olive in eleven cats gave a positive retrograde labelling of rostral rubral cells in only three animals, with a very few cells in each case. In contrast to this, olivary injections of horseradish peroxidase labelled lectin (wheat germ agglutinin) result in an abundance of retrogradely labelled cells within the rostral part of the ipsilateral red nucleus, the great majority of these cells being of the small and medium-sized type. Many of the retrogradely labelled rubral cells are only faintly stained. However, free horseradish peroxidase appears to be well suited as an anterograde tracer for a demonstration of the rubro-olivary tract. The findings are discussed in relation to the observation that in the cat only peroxidase conjugated to lectin can be used successfully as a retrograde tracer for a visualization of the rubro-olivary connection.

Conjugates of horseradish peroxidase (HRP) with cholera toxin and wheat germ agglutinin are superior to free HRP as orthogradely transported markers

The relative sensitivity of horseradish peroxidase (HRP) conjugates of cholera toxin (CTHRP) and wheat germ agglutinin (WGHRP) as orthogradely transported markers was compared with that of free HRP (FHRP) following unilateral, intravitreal injections of each probe in the rat. Both CTHRP and WGHRP labeled all of the recognized retinal efferents of the rat and were 30–50 times more sensitive than FHRP. In additional experiments the ‘receptor’ specificity of CTHRP and WGHRP was also demonstrated.

Visualization of binding and internalization of a horseradish peroxidase-hCG conjugate by monkey luteal cells.

We have prepared a conjugate of human chorionic gonadotropin (hCG) and horseradish peroxidase (HRP) that retained binding affinity for cell receptors and was biologically active. The hCG was covalently linked to dinitrophenylated HRP to produce a conjugate that contained HRP and hCG in a 1.6:1 molar ratio. The conjugate was separated from free hCG and HRP by gel chromatography. The isolated HRP;hCG had a biological activity that elicited dose-dependent enhancement of progesterone secretion by dispersed monkey luteal cells in vitro. The specificity of the conjugate to LH/hCG receptors was demonstrated by the insignificant nonspecific binding of HRP:hCG to dissociated cells that were previously saturated with native hCG. Electron microscopic studies revealed that the HRP:hCG was distributed uniformly on the surface of Iuteal cells previously fixed with aldehyde, and to some extent on the surface of those cells that were exposed to the conjugate at 4#{176}C. In those cells labeled at 4#{176}C, rinsed and warmed to 37#{176}C, a rapid redistribution of HRP:hCG occurred into small clusters. When dissociated cells were incubated with the hCG conjugate at 37#{176}C, it was localized in small clusters on the cell surface. Internalization of small aggregates of HRP:hCG occurred within 5 mm at 37#{176}C. This process occurred through invaginations of smooth segments of the plasma membrane. Once internalized, the conjugate was located within membrane bound vesicles of the cytoplasm. After 120 mm at 37#{176}C, the HRP:hCG was located in large cytoplasmic vesicles near the periphery of the cells, but they were not seen in association with Golgi complexes or lysosomes.

A light and electron microscopic study of the intraneuronal transport of horseradish peroxidase and wheat germ agglutinin-peroxidase conjugates in the rat visual system.

The ability of horseradish peroxidase (HRP) and the lectin wheat germ agglutinin (WGA) covalently conjugated with HRP to label retrogradely dorsal lateral geniculate nucleus (dLGN) neurons, subsequent to injections of either marker into rat striate cortex, was assessed by counting labelled neurons in the dLGN. Rats injected with either marker in concentrations ranging from 0.1 to 100 micrograms/microliter of HRP either free or coupled to WGA were perfused 24 h later and their brains incubated using the chromagen tetramethyl benzidine. At high concentrations (2-100 micrograms/microliters), comparable numbers of labelled neurons were observed in the dLGN but at low concentrations (0.1-1.0 micrograms/ microliters), WGA-HRP labelled 2-5 times more dLGN neurons than did unconjugated HRP. The sugar N-acetylglucosamine, and free WGA added in excess to WGA-HRP, abolished the retrograde labelling of dLGN neurons. In additional rats, which received striate cortex injections of 100 micrograms/microliters of either free HRP or HRP coupled to WGA, the injection site was studied with electron microscopy after survivals of 30 min to 24 h. Similar organelles in neuronal perikarya, dendrites and axons were labelled by both markers, with the exception that only rats injected with WGA-HRP had labelled GERL in some of their neurons in striate cortex. It was concluded from these studies that: (1) WGA-HRP is a more sensitive retrograde marker than free HRP at low concentrations in the rat visual system; (2) WGA-HRP binds specifically to moieties with terminal N-acetylglucosamine; and (3) WGA-HRP, but not free HRP, is localized to neuronal GERL of striate cortex subsequent to endocytosis.

A technique for preparing defined conjugates of horseradish peroxidase and immunoglobulin

Conjugates of horseradish peroxidase (HRP) and sheep anti-human immunoglobulin (anti-Ig) were prepared by means of a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The molar ratios of the conjugates could be changed by varying the degree of chemical substitution of anti-Ig and HRP. Gel filtration and affinity chromatography were used to separate conjugated anti-Ig from free HRP and unconjugated anti-Ig. The distribution of complex sizes and the presence of free HRP and anti-Ig in the final products were monitored by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. After final purification and characterisation conjugates were studied as reagents in enzyme immunoassays (EIA) with various antigens. Conjugates prepared by SPDP bridging were shown to yield more than 60% of the anti-Ig as conjugated small defined enzyme-protein complexes. A suitably labelled conjugate reacted in a standardised way resulting in a reproducible dose-response curve when a positive serum was assayed in different dilutions.

Pathways involved in fluid phase and adsorptive endocytosis in neuroblastoma.

The endocytosis of ricin, horseradish peroxidase (HRP), and a conjugate of ricin-HRP by monolayer cultures of murine neuroblastoma was studied using morphological and biochemical techniques. The binding of (125)I-ricin and (125)I-ricin-HRP to cells at 4 degrees C, as a function of ligand concentration, was a saturable process. The apparent affinity constants, determined at equilibrium, were 2.8 X 10(6) M(-1) for ricin and 1 x 10(6) M(-1) for ricin-HRP. The number of binding sites per cell was 8 x 10(7) and 3 x 10(7) for the lectin and the conjugate, respectively. The binding of (125)I-ricin to monolayers as not proportional to cell density. We found reduced binding at higher cell concentrations, suggesting a decrease in the accessibility of the ligand for the receptor site or fewer sites with increasing cell population. Neuroblastoma cells have an acid-phosphatase-positive network of cisternae and vesicles near the Golgi apparatus (GERL). Ricin-HRP undergoes endocytosis in vesicles and cisternae corresponding to GERL, and in residual bodies (dense bodies). The cellular uptake of ricin-HRP was 100-200 times greater than free HRP and there was no stimulation of fluid phase endocytosis by ricin. When monolayers were exposed to concentrations of native HRP 100-fold that of the conjugate, cellular uptake of peroxidase was comparable, but HRP was localized only in residual bodies and never in elements of GERL. These results support the conclusion that GERL is involved in the adsorptive endocytosis of ricin-HRP, while residual bodies are involved in the bulk uptake of HRP. In addition, the binding, uptake, and possible recycling of (125)I- subunit B (the binding subunit) of ricin and of (125)I-ricin was examined by quantitative electron microscope autoradiography. Both ricin and its binding subunit displayed similar autoradiographic grain distributions at 4 degrees C, and there was no evidence of their breakdown or recycling to the plasma membrane during endocytosis for 2 h.

Additional factors influencing sensitivity in the tetramethyl benzidine method for horseradish peroxidase neurohistochemistry.

In experiments that use horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) for tracing neural connections, the activity of tissue-bound enzyme as well as the stability of the resultant reaction product are influenced by the duration of storage, the composition of the storage medium, the type of counterstaining and even the details of histological dehydration. Furthermore, the conditions for preserving HRP activity are very different from those necessary for preserving the stability of the tetramethyl benzidine (TMB) reaction product. Thus, tissue-bound HRP activity is stable at a neutral pH, while a much lower pH, around 3.3, is required for preserving the stability of the TMB reaction product. Recent evidence indicates that the stabilization bath in sodium nitroferricyanide that was previously recommended is not necessary. However, gradual dehydration of mounted sections is essential for long-term stability. Excessive counterstaining and excessive dehydration interfere with the detection of reaction product. These considerations are pertinent to experiments using free HRP as well as to those where the enzyme has been conjugated to wheat germ agglutinin.

Intracellular transport in neurons.

Intracellular transport in neurons.

In vivo uptake of wheat germ agglutinin-horseradish peroxidase conjugates into neuronal gerl and lysosomes

In published studies we have shown that lectins and cholera toxin bind at 4°C on surfaces in cultured neurons and neuroblastoma cells and, after incubations of labeled cells at 37°C, undergo endocytosis (adsorptive endocytosis) into GERL (Golgi-endoplasmic reticulum-lysosome). In this study conjugates of wheat germ agglutinin (WGA) with horseradish peroxidase (HRP), or free HRP, were injected in rat submandibular glands or over superior cervical ganglia, and their uptake was examined by electron microscopic cytochemistry for HRP. Lysosomes and vesicles and cisternae of GERL were involved in the uptake of WGA-HRP injected into the submandibular gland or into the superior cervical ganglion; HRP, administered in a similar fashion, but in amounts 10–15-fold higher than WGA-HRP, underwent endocytosis into lysosomes. The present in vivo studies, as well as previous in vitro studies, indicate that neuronal GERL is part of the endocytic pathway of various ligands which bind on corresponding surface (plasma membrane) ‘receptors’.

The use of lectins and cholera toxin for the detection of surface carbohydrates of cultured neurons and neuroblastoma.

Conjugates of horseradish peroxidase with the lectins ricin (d-galactose), wheat germ agglutinin (N-acetylglucosamine), phytohemagglutinin (N-acetylgalactosamine), and with cholera toxin (GM1 ganglioside) were used for a cytochemical detection of corresponding termin al carbohydrates, or glycolipids on cell surfaces of cultured neurons and neuroblastoma cells. Cells were labeled at 4 degrees C with the above ligands and their adsorptive endocytosis was studied after incubations at 37 degrees C in a medium free of ligand. Peroxidase was detected by the method of Graham and Karnovsky (J. Histochem. Cytochem. 14:291, 1966). Lectins and cholera toxin underwent endocytosis in cisternae and vesicles of GERL (Golgi-Endoplasmic Reticulum-Lysosome). We suggest that GERL is the primary ercipieint of adsorptively endocytosed plasma membrane "receptor"-ligand complexes which are thus degraded or possibly reutilized (recycling). Wheat germ agglutinin-horseradish peroxidase conjugates used in vivo for studies of retrograde axonal transport were significantly more sensitive than free horseradish peroxidase.

Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport.

We have compared the retrograde axonal transport of horseradish peroxidase (HRP), to the retrograde transport of HRP conjugated with wheat germ agglutinin (WGA). Morphometric studies have shown that WGA-HRP conjugates were 40 times more sensitive than free HRP, in the tracing of retrograde connections from the rat submandibular gland to the superior cervical ganglion. Also, WGA-HRP was more sensitive than free HRP in the tracing of retrograde connections from the rat tongue to the hypoglossal nucleus. Our findings with WGA-HRP are consistent with the observations by Schwab et al. who reported (-125I) WGA is a highly sensitive retrograde tracer (Brain Research 152:145, 1978 (22)).

Localisation of proteins in coated micropinocytotic vesicles during transport across rabbit yolk sac endoderm.

Rabbit yolk sac splanchnopleur exposed in utero to IgG-HRP and IgG-ferritin conjugates, rabbit and bovine anti-HRP antibodies, free HRP, ferritin and human IgG, was examined ultrastructurally in an attempt to determine whether or not coated micropinocytotic vesicles are involved in selectively transporting immunoglobulins across yolk sac endodermal cells. Human, rabbit and bovine IgG-HRP conjugates, rabbit anti-HRP antibodies, free HRP and human IgG, all become localised in coated micropinocytotic vesicles. Differences were observed in that only human IgG and rabbit anti-HRP antibodies could be located in the intercellular space and bovine IgG-HRP conjugate could not be detected in coated micropinocytotic vesicles in confluence with the lateral and basal plasmalemma. Bovine anti-HRP anti-bodies, IgG-ferritin conjugates, and free ferritin, could not be observed in coated micropinocytotic vesicles. All proteins were detected in macropinocytotic vesicles, and dense bodies resembling phagolysosomes. Results are discussed in the light of a proposal that selection occurs at the cell surface during formation of coated micropinocytotic vesicles and is not linked to intracellular proteolysis.

DISTRIBUTION OF IMMUNE DEPOSITS IN THE SKIN OF REVERSED PASSIVE ARTHUS REACTION USING HORSERADISH PEROXIDASE AS ANTIGEN

ABSTRACTThe distribution of immune deposits in the skin of reversed passive Arthus reaction was investigated using horseradish peroxidase (HRP) as antigen. Free and non‐reacted HRP, after leaking from small vessels, spreads widely in connective tissue, in epidermis, and in hair follicles in a diffuse and homogeneous pattern. When a specific antibody is administered, HRP appears as granular deposits, which can be considered to be immune deposits, and they adhere to the tissues. When sufficient amounts of antibody is intracutaneously administered, these granular deposits are seen at the wall of venules or in the vicinity of the venules, but when a small amount of antibody is injected, the deposits are seen widely in the connective tissue and are apt to be accumulated at the basement membrane of the dermo‐epidermal junction and of hair follicles. Free HRP passes the basement membrane easily, but the immune complexes are considered unable to pass it and are deposited under it.