An Immunohistochemical Approach to Differentiate Hepatic Lipidosis from Hepatic Phospholipidosis in Rats

Abstract

Hepatocellular vacuolation can be a diagnostic challenge since cytoplasmic accumulations of various substances (lipid, water, phospholipids, glycogen, and plasma) can have a similar morphology. Cytoplasmic accumulation of phospholipids following administration of cationic amphiphilic drugs (CAD) can be particularly difficult to differentiate from nonphosphorylated lipid accumulations at the light microscopic level. Histochemical methods (Sudan Black, Oil Red-O, Nile Blue, etc.) can be used to identify both nonphosphorylated and/or phosphorylated lipid accumulations, but these techniques require non-paraffin-embedded tissue and are only moderately sensitive. Thus, electron microscopy is often utilized to achieve a definitive diagnosis based upon the characteristic morphologic features of phospholipid accumulations; however, this is a low throughput and labor intense procedure. In this report, we describe the use of immunohistochemical staining for LAMP-2 (a lysosome-associated protein) and adipophilin (a protein that forms the membrane around non-lysosomal lipid droplets) to differentiate phospholipidosis and lipidosis, respectively in the livers of rats. This staining procedure can be performed on formalin-fixed paraffin embedded tissues, is more sensitive than histochemistry, and easier to perform than ultrastructural evaluation.

Enhanced mitophagy in Sertoli cells of ethanol-treated rats: morphological evidence and clinical relevance

“…Immunohistochemical staining for LC3 and LAMP-2 and quantitative analysis Generally, the methods for immunohistochemical staining of LC3 and LAMP-2 were done according to the manufacturer's recommendations and reported studies (Obert et al 2007;Nahdi et al 2010). In brief, after deparaffinization, microwave antigen retrieval, blocking endogenous peroxidase, and serum blocking, the primary antibody was applied for 1 h at room temperature.…”

Non-proliferative and Proliferative Lesions of the Cardiovascular System of the Rat and Mouse

“…Increased intracellular concentrations of cleaved caspase 3 visualized with immunolabeling is one method to identify cells in the apoptotic pathway that can be very difficult to detect with routine H&E staining (Mikaelian et al 2010). Immunolabeling with Lamp2 allows recognition of lysosomal accumulation characteristic of phospholipidosis that might be an alternative to an ultrastructural examination (Obert et al 2007) (Fig. 7

Figure 7. Cardiomyocyte vacuolation as a fine cytoplasmic microvesiculation with hematoxylin and eosin (HE) staining.

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Figure 8. Lamp2 IHC allows characterization of the microvesiculation as increased lysosomes (due to phospholipidosis).

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Proliferative and Nonproliferative Lesions of the Rat and Mouse Male Reproductive System

“…When they do, the vacuolation is frequently restricted to a specific region (which will vary with the compound). If phospholipidosis is suspected, special procedures such as examination of plastic-embedded thin sections stained with toluidine blue; immunohistochemistry (IHC) using LAMP-2 (Obert et al 2007) or electron microscopy should be performed for confirmation. Epididymal phospholipidosis has been reported in rats and mice given spinosad (Stebbins et al 2002;Yano et al 2002), and in rats given a selective dopamine 3 antagonist (Rudmann et al 2004).…”