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.
Thomas
Martin Alexander
MA
Institute of Pharmacology and Toxicology, Ruprecht-Karls University of Heidelberg, 68169 Mannheim, Germany. Martin.Thomas@pharmtox.uni-heidelberg.de
Lemmer
Björn
B
D000818:Animals; D001921:Brain / Q000033:anatomy & histology / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006358:Hot Temperature; D006868:Hydrolysis; D007031:Hypothalamus / Q000033:anatomy & histology / Q000737:chemistry / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D017830:Octoxynol / Q000737:chemistry; D010121:Oxytocin / Q000032:analysis / Q000737:chemistry; D010286:Paraventricular Hypothalamic Nucleus / Q000033:anatomy & histology / Q000737:chemistry / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D044140:Receptor, Angiotensin, Type 1 / Q000032:analysis* / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*; D013495:Supraoptic Nucleus / Q000033:anatomy & histology / Q000737:chemistry / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods*; D014667:Vasopressins / Q000032:analysis / Q000737:chemistry
D003902:Detergents; D011108:Polymers; D044140:Receptor, Angiotensin, Type 1; D014667:Vasopressins; D005557:Formaldehyde; D010121:Oxytocin; D017830:Octoxynol; C003043:paraform