The use of heat-induced hydrolysis in immunohistochemistry on angiotensin II (AT1) receptors enhances the immunoreactivity in paraformaldehyde-fixed brain tissue of normotensive Sprague-Dawley rats.

The research on components of the renin-angiotensin system delivered a broad image of angiotensin II-binding sites. Especially, immunohistochemistry (IHC) provided an exact anatomical localization of the AT(1) receptor in the rat brain. Yet, controversial results between in vitro receptor autoradiography and IHC as well as between immunohistochemical studies using various antisera started a vehement discussion concerning specificity and cross-reactivity of these antisera. In particular the magnocellular subdivision of the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) provided controversial results on the localization of AT(1) receptors. Both areas are known for angiotensin II-induced release of vasopressin (VP) and oxytocin (OXT). To evaluate the significance of the appropriate method of antigen retrieval and its relevance for the detection of AT(1) receptors we performed IHC on AT(1) receptors in paraformaldehyde-fixed and paraffin-embedded brain tissue of Sprague-Dawley rats using either the detergent Triton X-100 or microwave oven heating. This study demonstrates that heat-induced hydrolysis enhances the quality and quantity of immunoreactivity (IR) in IHC on AT(1) receptors. In the organum vasculosum lamina terminalis and in the parvocellular subdivisions of the PVN we report a distribution of AT(1)-like-IR similar to that observed with other methods. However, in addition, we provide evidence that distinct AT(1)-like-IR is also localized in few magnocellular neurons of the PVN and in few parvocellular neurons of the dorsal SON but not in magnocellular neurons of the SON. Moreover, parallel IHC indicates that few magnocellular OXT- or VP-releasing neurons of the PVN as well as parvocellular OXT-releasing neurons of the SON do also contain AT(1) receptors.

HistoGreen: a new alternative to 3,3'-diaminobenzidine-tetrahydrochloride-dihydrate (DAB) as a peroxidase substrate in immunohistochemistry?

Abide its toxicity, 3,3'-diaminobenzidine-tetrahydrochloride-dihydrate (DAB) was the most potent marker for immunochemistry at the light and electron microscopic level in the last decades. Recently, a sensitive substrate for immunohistochemical staining methods and in-situ hybridization, HistoGreen, was developed for the use with peroxidase. In peroxidase reactions, HistoGreen delivers a green staining product which is suitable for permanent embedding without water. In contrast to DAB, HistoGreen is not toxic. To evaluate its usefulness, we performed comparative immunohistochemistry on angiotensin II (AT1)-receptors with DAB- and HistoGreen-staining on paraffin embedded slices of the rat brain at the light microscopic level. This also included counterstaining with Mayer's Hemalum and Nuclear Fast Red, respectively. We could demonstrate that HistoGreen delivers a coarsely grained label which is fast detectable in light microscopy. HistoGreen equals DAB in the exact localization of the immunoreaction to a large degree but its reaction product is considerably less stable in alcohol and water than DAB. In combination with Nuclear Fast Red, HistoGreen provides excellent imaging properties for the visualization and documentation of immunoreactive structures paired with an adequate demonstration of cellular details. Its tendency towards rapid over-staining as well as its low stability will restrict the use of HistoGreen in some areas of immunohistochemical research, yet the new chromogen represents an interesting alternative to DAB at the light microscopic level.

Localization of components of the renin-angiotensin system in the suprachiasmatic nucleus of normotensive Sprague-Dawley rats: part B. angiotensin II (AT1)-receptors, a light and electron microscopic study.

The dominant pacemaker of the mammalian circadian clock, located in the suprachiasmatic nucleus (SCN), is of special interest for many pharmacological, physiological and immunohistological studies on angiotensins and their receptors. Based on its role in the circadian modulation of blood pressure and vasopressin release, the distribution and function of the neuropeptide angiotensin II (ANG II) and its AT1-receptors (AT1) in the SCN became a target for several immunohistological studies. Though the distribution of ANG II and vasopressin in the SCN is well known at light microscopic level, detailed data concerning the AT1-receptor distribution in the SCN is missing. To confirm the mechanisms by which ANG II exerts its actions in the SCN, it is vital to understand how the brain renin-angiotensin system is organized at the cellular level, including the distribution of ANG II and the ANG II (AT1)-receptors as well as the protein-receptor complex. The current paper presents a light- and electron microscopic study on AT1-receptor-immunolabeling in the suprachiasmatic nucleus of normotensive Sprague-Dawley rats.

Localization of components of the renin-angiotensin system in the suprachiasmatic nucleus of normotensive Sprague-Dawley rats: part A. angiotensin I/II, a light and electron microscopic study.

The central pacemaker of the mammalian circadian clock, identified in the suprachiasmatic nucleus (SCN), is of special interest for many chronomedical studies on neuropeptides. Based on its role in the modulation of blood pressure and vasopressin release, the distribution and function of the neuropeptide angiotensin II (ANG II) in the SCN became a target for several immunohistological studies. At the light microscopic level, the distribution of ANG II in the SCN is well known, but detailed information about the localization of ANG II in the SCN at the ultrastructural level is missing. To gain further insight in the functional aspects of ANG II in the SCN, we investigated on the subcellular localization of the neuropeptide ANG II and its precursor ANG I in the SCN. The current report presents a light and electron microscopic study on ANG I/II-immunoreactivity in the suprachiasmatic nucleus of normotensive Sprague-Dawley rats.

Localization of angiotensin II (AT1)-receptor-immunoreactive fibres in the hypothalamus of rats: angiotensin II-sensitive tanycytes in the ependyma of the third ventricle?

Scanning the hypothalamus of rats for receptor binding sites of the octapeptide hormone angiotensin II (ANG II), we observed ANG II-sensitive fibres in the ventrolateral hypothalamus. The ANG II (AT(1))-receptor-immunoreactive processes originate from cells-probably tanycytes-embedded in the base and the ventrolateral walls of the third ventricle and reach into the retrochiasmatic area, the ventrolateral hypothalamus and the median eminence.

Subcellular identification of angiotensin I/II- and angiotensin II (AT1)-receptor-immunoreactivity in the central nervous system of rats.

To gain insight into generating and transport mechanisms of angiotensin (ANG) in the brain the study was focused on the subcellular localization of ANG II and its AT(1)-receptors in the hypothalamus of rats. The present paper demonstrates ANG II- and AT(1)-receptor-immunolabelling at brain parenchyma vessels and at glial and neuronal structures in the perivascular region. Further, ANG II- and AT(1)-receptor-immunoreactivity is shown at plasma membranes and intracellular structures in the ependyma of the third ventricle. Based upon a conventional horseradish peroxidase technique, combined with the classical substrate 3,3'-diaminobenzidine, a procedure is introduced that will be useful with a variety of antibodies used on glutar- and paraformaldehyde-fixed brain tissue. This technique enables a fast correlation between light and electron microscopical results and might also provide an attractive alternative to colloidal gold-labelling and silver-intensification techniques.