Detection of cellular hypoxia by pimonidazole adduct immunohistochemistry in kidney disease: Methodological pitfalls and their solution.

Detection of cellular hypoxia by pimonidazole adduct immunohistochemistry in kidney disease: Methodological pitfalls and their solution. Am J Physiol Renal Physiol. 2019 Jun 12;: Authors: Ow CPC, Ullah MM, Ngo JP, Sayakkarage A, Evans RG Abstract Pimonidazole adduct immunohistochemistry is one of the few available methods for assessing renal tissue hypoxia at the cellular level. It appears to be prone to artifactual false-positive staining under some circumstances. Here, we assessed the nature of this false-positive staining and, having determined how to avoid it, re-examined the nature of cellular hypoxia in rat models of kidney disease. When a mouse-derived anti-pimonidazole primary antibody was used, two types of staining were observed. Firstly, there was diffuse staining of the cytoplasm of tubular epithelial cells which was largely absent when the primary antibody was omitted from the incubation protocol or in tissues known not to contain pimonidazole adducts. Secondly, there was staining of the apical membranes of the tubular epithelial cells, debris within the lumen of renal tubules, and tubular casts. This latter staining was present even when the primary antibody was omitted from the incubation protocol. Such false-positive staining was particularly prominent in acutely injured kidneys. It could not be avoided by pre-incubation of sections with a mouse immunoglobulin G blocking reagent. Furthermore, pre-adsorption of the sec...
Source: Am J Physiol Renal P... - Category: Urology & Nephrology Authors: Tags: Am J Physiol Renal Physiol Source Type: research