Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry

  • A.M. Schläfli University of Bern, Switzerland.
  • S. Berezowska | sabina.berezowska@pathology.unibe.ch University of Bern, Switzerland.
  • O. Adams University of Bern, Switzerland.
  • R. Langer University of Bern, Switzerland.
  • M.P. Tschan University of Bern, Switzerland.

Abstract

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.

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Author Biographies

A.M. Schläfli, University of Bern
Department of Experimental Pathology and Graduate School for Cellular and Biomedical Sciences, Institute of Pathology
S. Berezowska, University of Bern
Department of Clinical Pathology and Translational Research Unit, Institute of Pathology
O. Adams, University of Bern
Department of Experimental Pathology, Translational Research Unit, Institute of Pathology and Graduate School for Cellular and Biomedical Science
R. Langer, University of Bern
Department of Clinical Pathology and Translational Research Unit, Institute of Pathology Institute of Pathology
M.P. Tschan, University of Bern
Department of Experimental Pathology and Graduate School for Cellular and Biomedical Sciences, Institute of Pathology,
Published
2015-05-05
Section
Technical Notes
Keywords:
Immunohistochemistry, immunofluorescence, formalin fixed paraffin embedded tissue, LC3B, p62, cancer.
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How to Cite
SchläfliA., Berezowska, S., Adams, O., Langer, R., & Tschan, M. (2015). Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry. European Journal of Histochemistry, 59(2). https://doi.org/10.4081/ejh.2015.2481