Immunohistochemical analysis of macroautophagy

Abstract

T ransmission 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.

When Tissue Antigens and Antibodies Get Along

“…In some cases, the gold standard method does not prove the presence of a protein but demonstrates a physiological activity closely associated with the given protein. An example is the validation by Martinet et al 228 of an assay to demonstrate autophagy-related proteins. The gold standard to detect autophagy is transmission electron microscopy (TEM).…”

Autophagy in Non-Alcoholic Fatty Liver Disease (NAFLD)

“…Increased colocalisation of LDs with lysosomal markers such as lysosomal-associated membrane protein 1 L"MP1 [31] or lysotracker [35] in fat-loaded cells was also demonstrated by immunoluor-escence microscopy. Similarly, immunohistochemical LC3"-positive dots were localised on the surface of LDs [36]. Immunogold staining of LC3 on transmission electron microscopy TEM slides conirmed the colocalisation of LC3 with LDs and implies a LD-regulating function of autophagy [31][32][33].…”

“…Data of liver biopsy represent a snapshot of a very dynamic process and cannot accurately discern between increased autophagy and decreased degradation of autophagosomes [30,36]. Furthermore, some markers need to be overexpressed for accurate identiication of autophagic structures [36]. Nonetheless, a small post-mortem study demonstrated decreased LC3 and increased p6 staining in relation with the degree of steatosis, suggesting decreased autophagy in more severe steatosis [37].…”

“…Data of liver biopsy represent a snapshot of a very dynamic process and cannot accurately discern between increased autophagy and decreased degradation of autophagosomes [30,36]. Furthermore, some markers need to be overexpressed for accurate identiication of autophagic structures [36].…”

“…around the central veins [36,37]. Findings in glutamine metabolism postulate a theoretic zonal distribution of autophagy [3 ].…”