A stereologic approach to estimate the number of immunogold-labeled molecules in cells of tubules.
OBJECTIVE
The quantity of molecules can be measured very precisely by molecular biological methods, but the capabilities of these are limited to measure only the total mass of tissue. For estimating the number of molecules at the cell level, it is necessary to combine an immunohistochemical protocol with designed-based principles of stereology at the level of electron microscopy (EM). This article focuses on the problems and practical solutions of fitting together immunohistochemistry, stereology, and electron microscopy for the estimation of the number of angiotensin II AT1 receptors in rat kidney arterioles.


METHODS
We performed the preembedding immunostaining of angiotensin II AT1 receptors using the silver-enhanced immunogold labeling system at EM level on serial sections of renal arterioles from 5 rats.


RESULTS
Using this method the number of molecules can be estimated along the renal arterioles separately on the cell's surface, in cytoplasm, in nucleus, or in any subcellular location.


CONCLUSIONS
For estimating the number of AT1 receptors, we designed a protocol that took into account the requirements for both immuno-EM and stereology. This method can be applied for estimating any molecule number in different types of cells in tubules.
Razga
Zsolt
Z
Electron Microscopy Laboratory, Pathology Department, University of Szeged, Hungary. razgazst@patho.szote.u-szeged.hu
Randel Nyengaard
Jens
J
D000818:Animals; D001160:Arterioles / Q000033:anatomy & histology*; D007150:Immunohistochemistry / Q000379:methods*; D007678:Kidney Glomerulus / Q000033:anatomy & histology* / Q000737:chemistry; D007684:Kidney Tubules / Q000098:blood supply* / Q000737:chemistry; D008297:Male; D008855:Microscopy, Electron, Scanning; D051381:Rats; D011920:Rats, Inbred WF; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry*
D044140:Receptor, Angiotensin, Type 1