RRC ID 19386
Author Martinet W, Schrijvers DM, Timmermans JP, Bult H, De Meyer GR.
Title Immunohistochemical analysis of macroautophagy: recommendations and limitations.
Journal Autophagy
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.
Volume 9(3)
Pages 386-402
Published 2013-3
DOI 10.4161/auto.22968
PII 22968
PMID 23242143
PMC PMC3590258
MeSH Animals Autophagy* Autophagy-Related Protein 7 Immunohistochemistry / methods* Liver / pathology Mice Mice, Knockout Microscopy, Electron, Transmission / methods Microtubule-Associated Proteins / genetics Microtubule-Associated Proteins / metabolism
IF 11.059
Times Cited 27
Mice GFP-LC3#53 (RBRC00806)