Immunohistochemical analysis of macroautophagy

Abstract

Transmission electron microscopy (TEM) is an indispensable standard method to monitor macroautophagy in tissue samples. Because TEM is time consuming and not suitable for daily routine, many groups try to identify macroautophagy in tissue by conventional immunohistochemistry. The aim of the present study was to evaluate whether immunohistochemical assessment of macroautophagy-related marker proteins such as LC3, ATG5, CTSD/cathepsin D, BECN1/Beclin 1 or SQSTM1/p62 is feasible and autophagy-specific. For this purpose, livers from starved mice were used as a model because hepatocytes are highly sensitive to autophagy induction. ATG7-deficient mouse livers served as negative control. Our findings indicate that unambiguous immunodetection of LC3 in paraffin-embedded tissue specimens was hampered due to low in situ levels of this protein. Maximum sensitivity could only be obtained using high-quality, isoform-specific antibodies, such as antibody 5F10, in combination with Envision+ signal amplification. Moreover, LC3 stains were optimal in neutral-buffered formalin-fixed tissue, immersed in citrate buffer during antigen retrieval. However, even when using this methodology, LC3 monitoring required overexpression of the protein, e.g., in GFP-LC3 transgenic mice. This was not only the case for the liver but also for other organs including heart, skeletal muscle, kidney and gut. Immunohistochemical detection of the autophagy-related proteins ATG5, CTSD or BECN1 is not recommendable for monitoring autophagy, due to lack of differential gene expression or doubtful specificity. SQSTM1 accumulated in autophagy-deficient liver, thus it is not a useful marker for tissue with autophagic activity. We conclude that TEM remains an indispensable technique for in situ evaluation of macroautophagy, particularly in clinical samples for which genetic manipulation or other in vitro techniques are not feasible.

Keywords: :

• immunohistochemistry
• liver
• autophagy
• LC3
• GFP-LC3
• Envision+

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors are indebted to Rita Van Den Bossche, Hermine Fret, Anne-Elise Van Hoydonck, Lieve Svensson, Francis Terloo and Dominique De Rijck for excellent technical assistance. The authors also thank Dr. Masaaki Komatsu (The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) for providing Atg7^F/F mice. This work was supported by the Fund for Scientific Research (FWO)-Flanders (projects G.0431.11N, G.0448.11N, G.0443.12N and G.0074.12N) and the University of Antwerp (BOF). The Tecnai G2 Spirit BioTwin TEM was purchased with support of the Hercules Foundation (Hercules Type 2: AUHA004).