Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents.

Tetramethyl benzidine (TMB) is a presumptively non-carcinogenic chromogen which yields a blue reaction-product at sites of horseradish peroxidase activity. Sixty-six distinct procedures were performed in rats and monkeys in order to determine the optimal incubation parameters for TMB. As a result, a procedure is recommended whose sensitivity greatly surpasses that of a previously described benzidine dihydrochloride method. Indeed, the sensitivity of this new method in demonstrating retrograde transport is markedly superior to that of the previously described benzidine dihydrochloride method. Furthermore, as a consequence of this enhanced sensitivity, many efferent connections of the injection site are also visualized. The injection site demonstrated by this TMB procedure is significantly larger than the one demonstrated when benzidine dihydrochloride or diaminobenzidine is used as a chromogen. Finally, this TMB procedure has been compared to two other TMB procedures and found to provide superior morphology and sensitivity.     

Sensitivity in horseradish peroxidase neurohistochemistry: a comparative and quantitative study of nine methods.

Nine currently available methods for HRP neurohistochemistry have been compared with each other on matching tissue sections from four rats and four rhesus monkeys. The nine methods investigated in this report are the diaminobenzidine (DAB) procedures of LaVail JH and LaVail MM (J Comp Neurol 157:303, 1974), of Adams JC (Neuroscience 2:141, 1977) and of Streit P and Reubi JC (Brain Res 126:530, 1977); the benzidine dihydrochloride (BDHC) procedures of Mesulam M-M (J Histochem Cytochem 24:1273, 1976) and of De Olmos J and Heimer L (Neurosci Lett 6:107, 1977); the o-dianisidine (O-D) procedure of De Olmos J (Exp Brain Res 29:541, 1977); the p-phenylenediamine dihydrochloride and pyrocatechol (PPD-PC) procedure of Hanker JS et al., (Histochem J 9:789, 1977) and the tetramethyl benzidine (TMB) procedures of Mesulam M-M (J Histochem Cytochem 26:106, 1978) and of De Olmos J et al. (J Comp Neurol 181:213, 1978). Quantitative comparisons were based on counts of retrogradely labeled perikarya. The extent of anterograde transport and the size of the injection site were also compared at a more qualitative level. The results indicate that one TMB procedure (Mesulam M-M, J Histochem Cytochem 26:106, 1978) is distinctly superior to each of the other eight procedures in the number of labeled perikarya that it can demonstrate. Furthermore, these differences are statistically significant at better than the 0.05 level of confidence. Differences in sensitivity are most evident when the perikarya contain small quantities of transported HRP. The same TMB method also demonstrates more anterograde transport and a larger injection site than all the other procedures. If less sensitive procedures are employed, afferent or efferent connections that are clearly demonstrated by this TMB procedure are either underestimated or completely overlooked. It is suggested that sensitivity in HRP neurohistochemistry is determined by multiple factors which include the method of fixation, post-fixation storage, the choice of chromogen, the incubation parameters, the type of HRP enzyme that is administered, and the postreaction treatment.     

Horseradish peroxidase as a tracer for capillary permeability studies

The permeability of capillaries was investigated utilizing an in vivo injection of horseradish peroxidase (HRP) and an in situ perfusion of a balanced salt solution containing HRP and lanthanum chloride. In the continuous capillaries of heart and muscle, HRP diffuses mainly through intercellular junctions, while in testicular capillaries, the transport is via micropinocytotic vesicles. The diffusion and micropinocytotic transport of HRP was demonstrated in both directions, i.e. from the capillary lumen to the interstitium and vice versa. Lanthanum can be used as a bidirectional inhibitor of micropinocytosis. The transport of HRP is then almost completely hindered in testicular capillaries. In heart muscle, the effect on HRP transport is not significant, due to second transport pathway, i.e. intercellular cleft passage.     

A method for measuring the amount of retrogradely transported HRP in the rat masseteric motoneuron using Mesulam's HRP histochemical protocol and an image processing system. An investigation of the effect of dopamine receptor antagonists on the retrograde transport of HRP

We developed a method for a determination of the amount of retrogradely transported HRP in the rat masseteric motoneuron using a modification of Mesulam's HRP histochemical protocol and an image processing system combined with a light microscope and a television camera. To test the validity and reproducibility of the new method, a quantitative comparison of the amount of dark blue granules of HRP-product in the cell body of masseteric motor neurons was performed between the right and left trigeminal motor nuclei of 70 rats, which resulted in no significant difference. An additional study used the method was made of the effects of administration of five dopamine receptor antagonists with different biochemical and pharmacologic properties on retrograde transport of HRP in the rat masseteric motoneuron. As a result, chlorpromazine, haloperidol, SCH 23390, and sulpiride significantly enhanced retrograde transport of HRP as against domperidone which showed no significant change in the transport. A possible regulatory system for retrograde transport of HRP in the masseteric motoneuron was discussed in relation to the action of the dopamine receptor.     

Cytochemical examination of the compartments involved in the transcellular transport of horseradish peroxidase in rat hepatocytes

Horseradish peroxidase (HRP, 10 mg/100 g body weight) was intravenously injected into rats in order to investigate the nature of the compartments involved in the transcellular transport of the protein through hepatocytes into bile. Double cytochemistry for HRP and the marker enzymes for cytoplasmic organelles was used. HRP was shown to be taken up by hepatocytes via vesicles at the sinusoidal surface, some of which were positive for 5'-nucleotidase activity. HRP was then found in the smooth-surfaced vesicles and tubules which were negative in 5'-nucleotidase, glucose 6-phosphatase, thiamine pyrophosphatase and acid phosphatase activity, suggesting that the tubular structures are neither the endoplasmic reticulum, the Golgi apparatus nor lysosomes. Biochemical studies revealed that the lead procedures used for the double cytochemistry did not inhibit the peroxidatic activity of HRP, and conversely that HRP did not interfere with the marker enzyme activity. Such cytochemical observations seemed to be supported by the observation that administration of monensin (3.5 mg/100 g) and chloroquine (5 mg/100 g), which markedly altered the structure of the Golgi apparatus and lysosomes, respectively, slightly altered the biliary excretion of HRP but not to a significant extent.     

Dynamics of the transganglionic movement of horseradish peroxidase in primary sensory neurons

Summary The dynamics of horseradish peroxidase (HRP) transport in primary sensory neurons were studied in rats by demonstration of the reaction product in spinal nerves, spinal ganglia, dorsal roots and in the spinal cord at different survival times after application of the enzyme to the transected sciatic nerve and to the spinal cord. Using tetramethylbenzidine as the chromogen according to Mesulam (1978), transganglionic transport of HRP was shown in both the disto-proximal direction after peripheral application, and proximo-distal direction after central application. Significant differences in staining intensity between the central and peripheral processes of primary sensory neurons were found after all survival times used in this study. After peripheral application the number of labeled axons and the staining intensity were higher in spinal nerves than in dorsal roots; an inverse situation occurred after central application. These differences as well as the time sequences in staining of different parts of primary sensory neurons suggest that HRP applied to a peripheral nerve and to the spinal cord, respectively, enters the perikarya of spinal ganglion cells in any case before continuing its movement in a cellulifugal direction. Lysosomal degradation of the major portion of the applied HRP is supposed. However, in the post-perikaryal portion of a considerable number of neurons HRP-transport still occurs to a varying extent, thus resulting in labeling of nerve endings. In some neurons a post-perikaryal transport could not be detected light microscopically. The transport rates differ: the calculated transport rate of disto-proximal, cellulipetal movement in the fastest transporting neurons was 7.5 mm/h, that of the disto-proximal cellulifugal movement 2.5 to 3 mm/h.This work was partly supported by the Hartmann Müller-Stiftung I want to thank Miss Regula Eichholzer for the technical assistance     

On the anatomical organization of the vagal nuclei

The neurons of origin of the right vagus and its components in both the monkey (Macaca fascicularis) and albino rats were localized by the retrograde transport of horseradish peroxidase (HRP) applied to the stomach wall, the vagal trunk and its recurrent laryngeal branch. An attempt was also made to localize the neurons forming the superior laryngeal nerve and those supplying the thoracic organs by a combination of operative procedures. The results showed that the stomach was innervated by neurons distributed throughout the entire rostrocaudal extent of the dorsal motor nucleus (DMN) on both sides of the brain stem. Neurons scattered throughout the entire extent of the DMN and nucleus ambiguus (NA) supplied the thoracic viscera. There did not appear to be any topographic arrangement in the DMN neurons supplying the abdominal and thoracic viscera as reported by other workers, and there was no clear evidence of crossing of vagal fibers in the monkey brain stem, though such crossing was seen in the rat brain stem. Both the superior and inferior ganglia of the vagus nerve were labeled following application of HRP to the vagal trunk. Neurons in the caudal part of the NA gave rise to fibers in the ipsilateral recurrent laryngeal nerve, at least on the right side. The neurons giving rise to the superior laryngeal nerve could not be delineated in this study. In all the experimental procedures described, the hypoglossal nucleus was labeled only after applying HRP to the hypoglossal nerve.     

Double-enzyme conjugates, producing an intermediate color, for simultaneous and direct detection of three different intracellular immunoglobulin determinants with only two enzymes

A new double-enzyme conjugate was synthesized by coupling alkaline phosphatase (AP) to horseradish peroxidase (HRP). After AP (blue) and subsequent HRP (red) cytochemistry, this new conjugate produced a stable intermediate-colored (violet) product. By coupling this double-enzyme conjugate to an antigen (trinitrophenyl, TNP) or an antibody (anti-mouse immunoglobulin G2a), anti-TNP or -IgG2a-producing cells could be demonstrated as violet cells in spleen sections. This led to the development of a rapid one-step incubation--two-step cytochemical procedure for simultaneous detection of three different determinants in a single tissue section. To demonstrate this novel triple staining method, we coupled three different antigens to, respectively, AP, HRP, and AP-HRP. When spleen sections of immunized animals were incubated with a mixture of these three antigen-enzyme conjugates, we could distinguish antibody-forming cells against each of these three antigens simultaneously as red (HRP), blue (AP), and violet (AP-HRP) cells. The simultaneous detection of three different classes of intracellular antibodies in a single section also proved to be possible with this method. With this study we provide a new direct method for detection of three different intracellular immunoglobulins after a one-step incubation and a two-step standard cytochemical procedure.     

Horseradish peroxidase catalyzed degradation of industrially important dyes.

Horseradish peroxidase (HRP) is known to degrade certain recalcitrant organic compounds such as phenol and substituted phenols. Here, for the first time we have shown HRP to be effective in degrading and precipitating industrially important azo dyes. For Remazol blue, the enzyme activity was found to be far better at pH 2.5 than at neutral pH. In addition, Remazol blue acts as a strong competitive inhibitor of HRP at neutral pH. Horseradish peroxidase shows broad substrate specificity toward a variety of azo dyes. Kinetic constants (K(m)(app) and V(max)(app)) for two different dyes have been determined. In addition to providing a systematic analysis of the potential of HRP in degradation of dyes, this study opens up a new area on exploration of commercial dyes as inhibitors of enzymes. 2001 John Wiley & Sons, Inc.     

HRP uptake by olfactory and vomeronasal receptor neurons: use as an indicator of incomplete lesions and relevance for non-volatile chemoreception

The transport of HRP (horseradish peroxidase) from the nasalcavity to the brain by intact olfactory receptor axons was usedto investigate the effectiveness of methods commonly used inbehavioral studies for deafferenting nasal chemoreceptor systems.The HRP experiments demonstrated that routine intranasal lavagewith zinc sulfate solution fails to destroy all olfactory receptorneurons in hamsters, in spite of the distinct behavioral deficitthat this treatment can cause in the male hamster. The intracranialdeafferentation of the accessory olfactory bulb by surgicalsection of the vomeronasal nerves was generally effective butthere was much incidental damage to main olfactory nerves thatwould probably not be detected without the HRP tracer. The distribution pattern of HRP molecules introduced into themammalian nasal cavity, as shown by the uptake of HRP by nasalchemoreceptors and its transport to the brain, was also usedto identify potential pathways for non-volatile stimulus moleculeswithin the nose. HRP reaction product was reliably detectedin the glomeruli of the main olfactory bulb after HRP was depositedat the nostril, demonstrating that nonvolatile materials, oncethey have entered the nasal cavity, can reach the main olfactoryreceptor neurons in the posterior nasal epithelium. Significantamounts of HRP reaction product were never observed in the accessoryolfactory bulbunlessa large dose of epinephrine had been givento activate the vomeronasal organ pumping mechanism, which drawssubstances into the vomeronasal organ lumen. Thus, it seemsthat stimulus access to vomeronasal receptor neurons is controlledindependently of access to main olfactory receptor neurons.     

Polarity in the transcytotic processing of apical and basal membrane-bound peroxidase-polylysine conjugates in MDCK cells.

A conjugate of horseradish peroxidase (HRP) to poly(L-lysine) (PLL) was used to characterize a non-lysosomal proteolytic compartment in the MDCK Strain I epithelial cell line. This compartment is expressed in a polar fashion, and is capable of degradation of the PLL moiety in the conjugate followed by release of HRP via a basal-to-apical, but not apical-to-basal, transcytotic pathway. This uptake, cleavage, and transport process appears to require approximately 2 hr, as there is a 2 hr lag-time between conjugate administration to the basal surface and HRP release to the apical medium. Monensin (10 microM) failed to inhibit this process, indicating that participation of the trans-Golgi network (TGN) in the trafficking of internalized conjugate is not the rate-determining step. Inhibition of HRP transport was found to be elicited by 50 micrograms/ml leupeptin, but only when applied to the basal surface. Brief trypsinization of either the basal or apical surfaces of cells preloaded with HRP conjugate showed no appreciable inhibitory effect on the apical release of HRP, indicating that an intracellular compartment rather than surface-bound enzymes is responsible for the degradation of the PLL moiety in the conjugate. Our results demonstrate the presence of an intracellular proteolytic compartment which is accessible in the basal-to-apical, but not apical-to-basal, transport pathway; and this compartment can be exploited for the transcytosis of membrane-bound molecules.     

A reliable method combining horseradish peroxidase histochemistry with immuno-beta-galactosidase staining

A sensitive combination of horseradish peroxidase (HRP) tracing and immunohistochemistry was used by Rye et al. [J Histochem Cytochem (1984) 32:1145] in a search for the origins of neurotransmitter- and neuromodulator-containing nerve fibers in brain. In this combination, peroxidase as a marker in immunohistochemistry was thought to yield a homogeneous brown immunoreaction product of diaminobenzidine, different from the black granular reaction product of retrogradely transported HRP, which is visualized by the tetramethylbenzidine (TMB) reaction and subsequent stabilization. A neuron that exhibits both kinds of reaction products in its cytoplasm in sections subjected to combination staining is referred to as a double-labeled cell. With a combined HRP and corticotropin-releasing factor (CRF) immunoperoxidase-antiperoxidase (PAP) method, the first set of experiments showed "false" double-labeled cells in the pyramidal cell layer of rat cerebral cortex, but only rarely in the subcortical areas, possibly because of the use of one enzyme system in two different histochemical procedures. This limitation of the double-staining technique prompted us to demonstrate an alternate combination of HRP tracing and immunohistochemistry in the second set of experiments by employing two previously described independent enzyme systems: HRP as a retrograde tracer and beta-galactosidase as a marker for immunohistochemical demonstration of CRF. A homogeneous blue reaction product indicated immuno-beta-galactosidase staining, and a granular black or brown reaction product labeled retrogradely transported HRP in double-labeled cells in subcortical regions. Neither double labeling nor "false" double labeling was seen in pyramidal cells of cerebral cortex. These findings suggest that application of two independent enzyme systems in a combined HRP and immunohistochemical method may be useful for investigating in origins of peptidergic fibers in brain when the combination of HRP histochemistry and the PAP method appears to be inappropriate.     

Transcellular processing of disulfide- and thioether-linked peroxidase--polylysine conjugates in cultured MDCK epithelial cells

Horseradish peroxidase (HRP) was conjugated to nondegradable polycationic poly(D-lysine) (PDL) through either a thioether (HRP-S-PDL) or a disulfide (HRP-SS-PDL) linkage. The binding and transcytosis of these conjugates was studied in Madin-Darby canine kidney (MDCK) cell monolayers grown on 3-microns microporous polycarbonate filters. Conjugation of HRP to PDL with both linkages markedly increased the binding of this protein onto the cell monolayers. However, an enhancement of the transcellular transport of HRP in both apical-to-basal and basal-to-apical directions was observed only in HRP-SS-PDL, but not in HRP-S-PDL. HRP-SS-PDL transport was inhibited by colchicine and by 4 degrees C incubation. The transport of 14C-sucrose was not affected by the presence of conjugates. These results indicate that the transport of the conjugate across the cell monolayers was due to a transcellular process rather than to any leakage of the cell junction caused by polycations. The disulfide linkage between HRP and PDL was cleaved rapidly at the basal and, to a lesser extent, at the apical surface of the cell. Neuraminidase treatment decreased the binding of the conjugates onto the cell surface, but did not decrease the transcellular transport, suggesting that not all surface-bound conjugates were available for transcytosis. These results demonstrate that disulfide linkages can be cleaved during transcytosis in MDCK cells. The cleavage, however, occurs mostly at the binding site on the cell surface, which may prevent the cellular uptake of the intact conjugate.     

Effect of monensin on ricin and fluid phase transport in polarized MDCK cells.

The effect of monensin on endocytosis, transcytosis, recycling and transport to the Golgi apparatus in filter-grown Madin-Darby canine kidney (MDCK) cells was investigated using 125I-labeled ricin as a marker for membrane transport, and horseradish peroxidase (HRP) as a marker for fluid phase transport. Monensin (10 microM) stimulated transcytosis of both markers about 3-fold in the basolateral to apical direction. Transcytosis of HRP in the opposite direction, apical to basolateral, was reduced to approximately 50% of the control by monensin, whereas that of ricin was slightly increased. Recycling of markers endocytosed from the apical surface was reduced in the presence of monensin and there was an increased accumulation of both ricin and HRP in the cells. Transport of ricin to the Golgi apparatus increased to the same extent as the increase in intracellular accumulation. No change in recycling or accumulation was observed with monensin when the markers were added basolaterally, but transport of ricin to the Golgi apparatus increased almost 3-fold. Our results indicate that basolateral to apical transcytosis is increased in the absence of low endosomal pH, and they suggest that apical to basolateral transcytosis of a membrane-bound marker (ricin) is affected by monensin differently from that of a fluid phase marker (HRP).     

Quantification of protein transcytosis in the human colon carcinoma cell line CaCo-2

The transepithelial absorption of food-type proteins has been shown to proceed by endocytosis along two functional pathways: a minor direct pathway allowing transport of intact protein and a major lysosomal degradative pathway. The human colon carcinoma cell line CaCo-2 grown on Millipore filters was used here further to characterize these pathways by measuring HRP transport across the cell monolayer in Ussing chambers. In the apical-basal direction, this transport mainly occurred along the degradative pathway and was inhibited at 4 degrees C (7.41 +/- 1.26 pmoles/h.cm2 vs. 27.40 +/- 8.91 at 37 degrees C). The amount conveyed via the direct pathway was very small (0.89 +/- 0.35 pmoles/h.cm2) and did not diminish at 4 degrees C (1.43 +/- 0.59 pmoles/h.cm2). In the basal-apical direction, HRP transport along the degradative pathway at 37 degrees C was similar to the apical-basal value and was inhibited at 4 degrees C (16.40 +/- 4.05 vs. 2.72 +/- 2.52 pmoles/h.cm2), but along the direct pathway, it was eight times the apical-basal value (8.36 +/- 3.11 pmoles/h.cm2) and was inhibited at 4 degrees C (2.43 +/- 0.78 pmoles/h.cm2). Intact HRP fluxes were not correlated with the electrical resistance of the filters, indicating transport via a transcellular route. Monensin at 10(-5) M did not affect direct or degradative transport in the apical-to-basal direction. These results suggest that in CaCo-2 cells HRP undergoes bidirectional transcytosis by a fluid-phase mechanism, but the extent of degradation during that transport varies according to the membrane (apical or basal) where it is presented.     

Protein conjugation with amphiphilic block copolymers for enhanced cellular delivery

Modification of a model protein, horseradish peroxidase (HRP), with amphiphilic block copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic), was previously shown to enhance the transport of this protein across the blood-brain barrier in vivo and brain microvessel endothelial cells in vitro. This work develops procedures for synthesis and characterization of HRP with Pluronic copolymers, having different lengths of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks. Four monoamine Pluronic derivatives (L81, P85, L121, P123) were synthesized and successfully conjugated to a model protein, HRP, via biodegradable or nondegradable linkers (dithiobis(succinimidyl propionate) (DSP), dimethyl 3,3'-dithiobispropionimidate (DTBP), and disuccinimidyl propionate (DSS)). The conjugation was confirmed by HRP amino group titration, matrix-assisted laser desorption/ionization-time of flight spectroscopy, and cation-exchange chromatography. HRP conjugates containing an average of one to two Pluronic moieties and retaining in most cases over 70% of the activity were synthesized. Increased cellular uptake of these conjugates was demonstrated using the Mardin-Derby canine kidney cell line and primary bovine brain microvessel endothelial cells. The optimal modifications included Pluronic L81 and P85. These copolymers have shorter PPO chains compared to Pluronic P123 and L121, which were less efficient. There was little if any dependence of the uptake on the length of the hydrophilic PEO block for the optimal modifications. The proposed modifications may be used to increase cellular uptake of other proteins.     

Direct electrochemistry and electrocatalysis based on film of horseradish peroxidase intercalated into layered titanate nano-sheets

Intercalation of horseradish peroxidase (HRP) into layered titanate by assembling it with titanate nano-sheets (TNS) was firstly used for fabrication of enzyme electrode (HRP-TNS electrode). XRD result revealed that HRP-TNS film featured layered structure with HRP monolayer intercalated between the titanate layers. UV-vis spectra result indicated the intercalated HRP in TNS film well retained its native structure. The HRP-TNS film was uniform with porous structures which were confirmed by SEM. The immobilized HRP in the TNS film exhibited fast direct electron transfer and showed a good electrocatalytic performance to H2O2 with high sensitivity, wide linear range and low detection. The excellent electrochemical performance of the HRP-TNS electrode was attributed to biocompatibility of the titanate sheets, porous architectures of the HRP-TNS film which retained activity of HRP to large extent, avoided aggregation of HRP, provided better mass transport and allowed more HRP loading per unit area. Thus, the simple method described here provides a novel and effective platform for immobilization of enzyme in realizing direct electrochemistry and has a promising application in fabrication of the third-generation electrochemical biosensors.     

戊巴比妥对大鼠骨胳肌辣根过氧化物酶(HRP)的轴突摄取和逆行传送作用

腓肠肌内注射HRP后,用生物化学法测定坐骨神经、L_(4-6)节段背根和腹根神经的HRP含量。在戊巴比妥连续全身麻醉大鼠的HRP含量明显低于不麻醉的大鼠,而肌肉不活动(TTX中毒和切腱)大鼠神经组织中的HRP含量无甚变化。刺激神经不能改变麻醉大鼠的HRP含量。上述结果提示:除麻醉剂造成的肌肉不活动因素外,戊巴比妥对大鼠骨胳肌HRP的轴突摄取和逆行传送具有另外的抑制作用。已有研究报道:破伤风和单纯性疱疹病毒脑炎都是由于它们的毒素或病毒,通过外周神经摄取然后逆行传送到各级中枢而致病的。     

Electron microscopic demonstration of neural connections using horseradish peroxidase: a comparison of the tetramethylbenzidine procedure with seven other histochemical methods

Eight methods for the electron microscopic demonstration of horseradish peroxidase (HRP) labeling have been compared in adjacent series of vibratome sections of mouse lumbar spinal cord. The tracer, a HRP-wheat germ agglutinin (WGA) conjugate, was injected into the gastrocnemius muscle complex. Following retrograde axonal transport to the lumbar motor neurons and transganglionic anterograde transport of the tracer to the dorsal horn, the HRP activity was demonstrated in eight series of adjacent sections of lumbar spinal cord using eight methods. These included procedures using tetramethylbenzidine (TMB), benzidine dihydrochloride (BDHC), o-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and 4 methods using 3,3'-diaminobenzidine (DAB). All eight methods were able to demonstrate both retrograde labeling of motor neurons and transganglionic anterograde transport into the dorsal horn. However, there were differences in the appearance of the various reaction products under the electron microscope. In addition, differences in the distribution of the reaction products were observed by both light and electron microscopy. The largest distribution of reaction product was observed with TMB. BDHC and o-tolidine were next, followed by the DAB procedures and PPD-PC. The TMB, BDHC, and o-tolidine reaction products were all found to be suitable for electron microscopy. The TMB reaction product was electron dense and had a very distinctive crystalloid appearance that made identification of HRP-labeled neuronal profiles easy and unequivocal.     

Chromogenic substrates for horseradish peroxidase

Two new detection systems for horseradish peroxidase (HRP) have been developed for the staining of membranes used in immunoassays. These systems use dimethyl or diethyl analogues of p-phenylenediamine with 4-chloro-1-naphthol to generate a blue product or 3-methyl-2-benzothiazolinone hydrazone with 4-chloro-1-naphthol to generate a red product. These reagents offer increased sensitivity and lower background staining than currently available chromogenic detection substrates. In addition, the incorporation of these substrates increases the sensitivity of HRP labels to be comparable to that of alkaline phosphatase with the 5-bromo-4-chloro-3-indolyl phosphate + nitro blue tetrazolium substrate.