Original Article doi: 10.5146/tjpath.2017.01405 A Unique Immunofluorescence Protocol to Detect Protein Expression in Vascular Tissues: Tacking a Long Standing Pathological Hitch Puneet GANDHI, Richa KHARE Department of Research, Bhopal Memorial Hospital & Research Centre, BHOPal-38, INDIA ABSTRACT Objective: Autofluorescence induced interference is one of the major drawbacks in immunofluorescence analysis of formalin-fixed paraffin-embedded tissues, as it decreases the signal-to-noise ratio of specific labeling. Apart from aldehyde-fixation induced artifacts; collagen and elastin, red blood cells and endogenous fluorescent pigment lipofuscin are prime sources of autofluorescence in vascular and aging tissues. We describe herein, an optimized indirect-immunofluorescence method for archival formalin-fixed paraffin-embedded tissues tissues and cryo sections, using a combination of 3-reagents in a specific order, to achieve optimal fluorescence signals and imaging. Material and Method: Human telomerase reverse transcriptase, a protein implicated as a proliferation marker, was chosen relevant to its expression in solid tumors along with 3 other intracellular proteins exhibiting nuclear and/or cytoplasmic expression. Staining was performed on 10 glioma tissue sections along with 5 of their cryo sections, 5 sections each of hepatocellular, lung, papillary-thyroid and renal cell carcinoma, with 10 non-malignant brain tissue samples serving as control. Specimens were imaged using epifluorescence microscopy, followed by software-based quantification of fluorescence signals for statistical analysis and validation. Results: We observed that the combined application of sodium-borohydride followed by crystal violet before antigen retrieval and a Sudan black B treatment after secondary antibody application proved to be most efficacious for masking autofluorescence/non-specific background in vascular tissues. Conclusion: This unique trio-methodology provides quantifiable observations with maximized fluorescence signal intensity of the target protein for longer retention time of the signal even after prolonged storage. The results can be extrapolated to other human tissues for different protein targets. Key Words: Autofluorescence, Immunofluorescence, Immunohistochemistry, Signal intensity INTRODUCTION localization by immunofluorescence, making quantifiable assessment difficult and therefore inherently subjective (4). Immunofluorescence (IF) is a sensitive and versatile tool for histological studies (1). However, IF staining of formalin- Autofluorescence induced interference is one of the major fixed- paraffin-embedded (FFPE) tissues is not a routinely hitches in IF analysis of FFPE tissues, as it decreases the used method in clinical pathology due to the associated signal-to-noise ratio of specific labeling. The greatest source autofluorescence. On the other hand, immunoperoxidase of autofluorescence in mammalian tissues is artifacts due staining of FFPE tissues is a fundamental technique for to aldehyde fixation along with endogenous fluorophores: diagnostic pathology as well as research, although there are collagen, elastin, lipofuscin, red blood cells, and others. some major limitations associated with it. The first among Vascular tissues contain massive amounts of collagen and these is the resolution of antigen localization by light elastin, along with haem, which are typical constituents of microscopy that remains limited because chromogenic blood vessels (5-7), while lipofuscin is known to accumulate substrate saturates and precipitates easily. Another issue is with aging and oxidative stress in various tissues (8,9). the thickness of the sections that are imaged for analysis Under a fluorescence or confocal microscope, lipofuscin (2). Sometimes the antigen diffuses prior to fixation, appears as tiny, dot like structures within the cells, strongly making localization difficult specially in the case of soluble fluorescing over a wide excitation range of 360-647 nm (10), proteins like Glial Fibrilliary Acidic Protein (GFAP) (3). which overlaps with the commonly used fluorophores and All of these conditions hinder evaluation. Additionally, the interferes with specific immuno-fluorescent signals (11). In conventional staining method is less specific than antigen the present method validation, we describe the optimized (Turk Patoloji Derg 2018, 34:57-65) Correspondence: Puneet GANDHI Bhopal Memorial Hospital & Research Centre, Received : 21.03.2017 Accepted : 14.06.2017 Department of Research, BHOPAL, INdiA E-mail: [email protected] Phone: +91 755 274 21 52 57 Turkish Journal of Pathology GANDHI P and KHARE R: Unique IF Method for Protein Expression indirect IF method on vascular, archival FFPE tissues and each. The slides were then dipped in 100% and 95% ethanol cryo sections. for 2 changes of 5 minutes each, followed by immersing Glioma (brain tumor), being highly vascular, proliferative in 80%, 70% and 50% alcohol respectively, for 5 minutes and invasive, was considered for the present study. The each. Finally, slides were rinsed in running tap water for 15 technique was also performed on brain cryo-sections minutes. and non-malignant brain tissue sections along with lung, Pre-Treatment kidney and thyroid tissue sections and their results were Freshly prepared 0.1% sodium borohydride in phosphate compared for reproducibility. Expression of protein human buffer saline (PBS, pH 7.4) was applied to sections for 2 Telomerase Reverse Transcriptase (hTERT), implicated as changes, 2.5 minutes each without any in-between wash. a proliferation marker in various cancers (12-14), was used to verify and replicate the procedure, and quantitatively Then 0.5% crystal violet (10% stock in methanol) freshly analyze and statistically validate the methodology using diluted in normal saline was laid on the sections for 9 fluorescence tagged antibody. Subsequently, expression of minutes and drained. Lastly, 0.9% NaCl was overlaid on the other nuclear and cytoplasmic fluorescein isothiocyanate tissue, creating a bubble of liquid over the section for three (FITC) tagged protein targets, namely High Mobility washes of 5 minutes each. GroupA1 (HMGA1), Kinesin Family Member14 (KIF-14) Epitope Retrieval and Synaptophysin, was also checked on glioma tissues. Sodium citrate buffer (0.01 M, pH=6) was preheated at MATERIAL and METHODS P-high (1350 watt) for 5 minutes, in a microwave oven. Treatment Reagents The slides were immersed in this pre-warmed retrieval solution and the sections boiled for 6 minutes at P-high. The following reagents were used in this study: Neutral After a pause of 1 minute to lower down the temperature of detergent (Extran, Merck, UK), acetic acid (Merck, UK), retrieval solution, the sections were re-heated at P-40 (540 ethanol (Merck, UK), poly L-lysine (Sigma, USA), sodium watt) for 9 minutes. The container was removed to room borohydride (Hi-media, India), crystal violet (Hi-media, temperature and the slides allowed to cool for 20 minutes. India), NaCl (Rankem, India), sodium citrate (Qualigens, On cooling, the slides were flushed in running tap water for India), Phosphate buffer saline (PBS) (Thermo fisher 15 minutes. Excess water was drained-off from the slides Scientific, India), Tris (hydroxymethylaminomethane- and the sections covered with PBS (pH=7.4), creating a Qualigens, India), Triton X-100 (Sigma, USA), horse bubble of buffer over the tissue for 5 minutes. serum (Santa Cruz Biotechnology, USA), BSA (bovine serum albumin- Sigma, USA), hTERT primary antibody Troubleshoot (Monoclonal, Abcam, UK), HMGA1 (Monoclonal, Standardization of protocol was done by immersing the Abcam, UK), Synaptophysin (Monoclonal, Santa Cruz slides directly in retrieval solution and boiling the sections Biotechnology, USA), KIF 14 (Monoclonal, Santa for 5 minutes at P-high, P-80 (1080 watt), P-60 (810 watt) Cruz Biotechnology, USA), host specific Fluorescein and P-40, which did not give satisfactory florescence signal isothiocyanate (FITC) labeled secondary antibodies (Santa intensity. Cruz Biotechnology, USA), 4’, 6-Diamidino-2-phenylindol (DAPI, Hi-Media, India), p-phenylenediamine (Anti-fade, Permeabilization Sigma, USA), Sudan black B (SBB, Hi-Media, India). The sections were permeabilized by dipping in 0.2% Triton The specific labelling of hTERT protein in selected tissue- X-100 in PBS (pH 7.4) for 45 minutes at room temperature. sections by immuno-florescence immunohistochemistry Slides were rinsed and PBST (0.1% Tween in PBS) dropped (IF-IHC) was performed as per the protocol discussed here. on the tissue, creating a layer of buffer over the section for To start with, slides were cleaned with neutral detergent, 5 minutes. washed in running tap water for 30 min, and further dipped Troubleshoot in 1% acidic ethanol followed by rinsing in deionised water. The slides were then coated with poly-L-lysine solution • 1% Triton X-100 in PBS (pH 7.4) for 30 minutes at room (1:10, diluted in deionised water), then kept overnight at temperature room temperature. 3 µm sections were overlaid onto the • 0.5% Triton X-100 in PBS (pH 7.4) for 45 minutes at coated slides and coded. At the beginning of the procedure, room temperature tissue sections were deparaffinized in the oven at 65°C, followed by immersing in 3 changes of xylene, 5 minutes Both the above combinations did not yield desired results. 58 Vol. 34, No. 1, 2018; Page 57-65 GANDHI P and KHARE R: Unique IF Method for Protein Expression Turkish Journal of Pathology Blocking Mounting 70 µl blocking