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

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 nonneoplastic 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.

Prognostic value of the autophagy markers LC3 and p62/SQSTM1 in early-stage non-small cell lung cancer

“…For the purposes of correlation with pathological and clinical parameters, immunohistochemistry scores were categorized as either “low” or “high” for each staining pattern according to our established protocol [24] with slight modifications, following the prognostic impact of the single scores: For LC3 dot-like staining scores 0 and 1 were classified as low, scores 2 and 3 as high. For p62 dot-like staining score 0 was classified as low, scores 1, 2 and 3 were interpreted as high.…”

“…We investigated a homogeneous, large early-stage NSCLC cohort of 466 patients for the expression of the autophagy markers LC3 and p62, using a previously validated immunohistochemical protocol [24]. The observed differential expression of both markers points towards a biologically significant role in NSCLC, although drawing conclusions from the staining patterns on specific disruptions of the autophagy mechanism in the tumors is not clear-cut.…”

“…All samples were incubated with the following primary antibodies for 30 minutes at room temperature (RT), as described before [24]: LC3B from Cell Signaling Technology (MA, USA, #3868, clone D11, dilution 1:3000), LC3 from Novus Biologicals (Cambridge, UK, #NB600-1384, dilution 1:3000) using Tris buffer (pH 9) at 95°C for 30 minutes for antigen retrieval, p62 from MBL (IL, USA, #PM045, dilution 1:8000) using Citrate buffer (pH 6.5) at 100°C for 30 minutes for antigen retrieval. Antibody detection was done with the Bond Polymer Refine Detection kit (Leica Biosystems, DS9800) following the manufacturer's instructions.…”

Knockdown of Linc00515 Inhibits Multiple Myeloma Autophagy and Chemoresistance by Upregulating miR-140-5p and Downregulating ATG14

“…ATG7 also mediates ATG5-ATG12-ATG16 complex formation and the latter along with LC3-II is highly critical for autophagosome formation [17,18]. Adaptor protein p62/SQSTM1 binds to ubiquitinated proteins and LC3-II for mediating autophagy via localizing into autophagic compartments, transporting ubiquitinated proteins and organelles for degradation [19]. Based on these properties of LC3-I, LC3-II and p62, those were used to measure autophagic flux under certain conditions.…”

Histochemistry today: Detection and location of single molecules

“…76,77 Several articles have recently been published in the European Journal of Histochemistry on new methods or novel applications of well-established microscopical or histochemical techniques. Some of them were intended to improve fixation, embedding and antigen-retrieval procedures for protein immunodetection, with an obvious interest for the techniques suitable for aldehyde-fixed/paraffin-embedded samples, [82][83][84][85][86] which allows to extend the use of histochemical reactions to archived samples. A group of papers [90][91][92][93] were focused on the acquisition and interpretation of autofluorescence spectra from unprocessed biological tissues in vivo and ex vivo.…”