Practical molecular biology: PROTEINS Prof. Dr. Julia Kzhyshkowska PD Dr. Alexei Gratchev Prof. Dr. W. Kaminski

Protein analysis in tissues Principles of protein detection Immunohistochemistry (IHC) Immunofluorescence (IF)

Protein Problem Molecular Weight (MW); how many forms; charge and shape; Posttranslational modifications; Biosynthetic pathways, half-life, degradation pathways; Intracellular localisation; trafficking pathways Integration in protein-protein networks; interaction with DNA, RNA, lipids; Expression profile in cells and tissues; Biological function: one or many, regulated or constitutive, intracellular or extracellular, ubiquitous or cell-type specific; Proteins in pathology: biomarkers and their role in the molecular mechanism of a disease; Therapeutic protein targeting 3

Protein identification Direct sequencing (for purified protein); MW: motility in the gel (usually for denatured proteins) or gel-filtration chromatography (usually for native proteins). These methods can be used for purified protein or protein complex with limited amount of components; Proteomics-based approaches for complex protein mixtures, for example serum samples or cell lysates Immunological detection (for purified proteins, protein complexes and crude material like cell lysates or tissue extracts) Enzymatic activity

Protein quantification Photometric detection: total protein amount in the sample ELISA: measurement of particular protein concentration Enzymatic reaction: quantification of activity, not the protein FACS: relative quantification of protein amount in the cell Western blotting, IHC, IF - semi-quantitative or qualitative

Immunological detection of the protein Methods: Immunohistochemistry (IHC), Immunofluorescence (IF), Enzyime-Linked Immunosorbent Assay (ELISA) Western Blotting (WB), Immunoprecipitation (IP), Fluorescence Activated Cell Sorting (FACS) Principle of recognition primary antibody binds to specific epitope (one or several) in the protein Principle of detection primary antibody or secondary antibody that recognise primary antibody is labelled (examples: HRP for IHC and Western blotting, fluorescent dye for IF and FACS)

Material for IHC and IF Paraffin embedded Fresh or frozen Tissue sections Fresh or frozen Tissue sections; Cells grown on cover slips; Cells sedimented on object glass using cytospin centrifuge Advantages Disadvantages Advantages Disadvantages Antigens are in a good shape, and most of primary antibodies can be used Intracellular localization studies are possible even in tissue sections Limited time of storage Retrospective analysis is not possible Extremely long storage time, Retrospective analysis can be done on archive material Antigen-retrieval has to be designed individually for most of antigens Only limited number of labeled primary antibodies recognize retrieved antigen

Fixation of fresh and frozen material Method of fixation has to be selected according to 1) the experimental task. Examples: For simple identification of the protein in the cell: acetone fixation is sufficient For precise identification of protein localization in the intracellular compartment PFA/triton is optimal 2) ability of the antibody to recognize fixed antigen. Most of antibodies recognize antigens only in specific conditions. Example from our lab: MS-1 antibody recognizes stabilin-1 in acetone-fixed cells, but not in PFA fixed cells Methanol-Acetone Fixation Fix in cooled methanol, 10 minutes at –20 °C. Remove excess methanol. Permeabilize with cooled acetone for 1 minute at –20 °C. Or Paraformaldehyde-Triton Fixation Fix in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 2-10 minutes. Paraformaldehyde-Methanol Fixation Permeabilize with cooled methanol for 5-10 minutes at –20 °C. PEM-Ethanol Fixation Fix in PEM buffer for 10 minutes. Rinse twice, briefly, with PBS. Permeabilize with cooled ethanol for 5-10 minutes at –20 °C

IHC and IF: overlapping terms Direct Indirect or enzyme or enzyme Advantages Disadvantages Advantages Disadvantages Cheap Fast Only limited number of labeled primary antibodies are available commercially Wide range of labeled secondary antibodies are available commercially It is always possible to design combination for double and triple staining Takes more time, sometimes is more expensive Additional control for the background staining is absolutely necessary 9

Controls 1. Antibody-independent of non-specific signals IHC IF Background signal coming from substrate Auto fluorescence 2. Antibody-dependent non-specific signals/cross-reactions for IHC and IF: 2.1 Non-specific signal coming from antibody alone Solution: optimization of concentration of secondary antibody (not signal has to be observed when primary antibody is not applied) 2.2 Non-specific signal coming from primary antibody. Following controls for primary antibody have to be used and concentrations have to be optimized: Isotype control for monoclonal antibody Preimmune serum for polyclonal antibody-containing serum Matching Ig for purified polyclonal antibody Important note: by optimization working concentrations has to be calculated NOT dilution

Experimental design for IHC 1 2 3 4 Substrate (DAB) for IHC + 1st antibody _ + Preimmune serum for polyclonal ab or isotype control ab for monoclonal ab Antigen- specific ab 2nd antibody

IHC and IF on frozen tissues: human lymph node samples Martens, Kzhyshkowska et al, J Pathology, 2006 12

Identification of double positive cells by IF Martens, Kzhyshkowska et al, J Pathology, 2006

IHC on frozen tissues: mouse tumour sections Amount of stabilin-1+ TAM is significantly decreased in TS/A-SI-CLP tumors compared to TS/A-vector tumors Schuiping Yin, TMR Student 2011/2012 Master Thesis

IF on frozen tissues of mouse tumor Analysis of co-expression of CD206 and stabilin-1 in TS/A-vector and TS/A-SI-CLP tumor Stabilin-1-CD206+ and stabilin-1+CD206+ TAM appear in TS/A-vector and TS/A-SI-CLP tumors. The main phenotype of TAM in TS/A-vector tumor is stabilin-1+CD206+, while the main TAM phenotype in TS/A-SI-CLP tumor is stabilin-1-CD206+ stabilin-1+CD206+ stabilin-1-CD206+ stabilin-1-CD206+ stabilin-1+CD206+ Schuiping Yin, TMR Student 2011/2012 Master Thesis stabilin-1+CD206+

Additional treatments are needed for staining of paraffin-embedded tissues 1. Deparaffinisation 2. Antigen retrieval

Deparaffinization Before proceeding with the staining protocol, the slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section. Protocol Place the slides in a rack, and perform the following washes: Xylene: 2 x 3 min Xylene 1:1 with 100% ethanol: 3 min 100% ethanol: 2 x 3 min 95% ethanol: 3 min 70 % ethanol: 3 min 50 % ethanol: 3 min Running cold tap water to rinse Keep the slides in the tap water until ready to perform antigen retrieval. At no time from this point onwards should the slides be allowed to dry. Drying out will cause non-specific antibody binding and therefore high background staining

Antigen retrieval The demonstration of many antigens can be significantly improved by the pre-treatment with the antigen retrieval reagents that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites. The techniques involved the application of heat for varying lengths of time to formalin-fixed, paraffin-embedded tissue sections in an aqueous solution (retrieval solution). This is called "Heat Induced Epitope Retrieval (HIER)". Another method uses enzyme digestion and is called "Proteolytic Induced Epitope Retrieval (PIER)".  HIER PIER Citrate buffer pH 6.0 Tris-EDTA buffer pH 9.0 EDTA buffer pH 8.0 Proteinase K Trypsin Chymotrypsin Pepsin Pronase

IHC staining of paraffin-embedded human breast cancer Aida Avdic, TMR Student 2011/2012 Master Thesis Expression of CD68 and stabilin-1 in breast cancer by the stages: stage I CD68 (A1 and A2); stage II CD68 (B1 an B2); stage III CD68 (C1 and C2); stage IV CD68 (D1 and D2); stage I stabilin-1 (E1 and E2), stage II stabilin-1(F1 and F2); stage III stabilin-1(G1 and G2); stage IV stabilin-1 (H1 and H2). Scale bars 100 μm (A1 to H1), 50 μm (A2 to H2).

Immunofluorescence/confocal microscopy on of paraffin-embedded human breast cancer Analysis of CD68+/stabilin-1+ macrophages Aida Avdic, TMR Student 2011/2012 Master Thesis

Multiple IF Most frequently double and triple IF are used Color code Ab CLEVER Ab F4 Merge Human placenta Stabilin-1 staining PL-FITC Stabilin-1 TGN-46 Merge Human macrophage Color code Red + green = yellow Red + blue = pink Green + blue = cyan Green + red + blue = white Most frequently double and triple IF are used Kzhyshkowska et al, JI, 2008 21

Multuple IHC Multiple staining can also be done with enzyme conjugated antibodies developed with different chromogen substrates to produce the end products of different colors

IHC: principle of EnVision detection system from DAKO Enzyme: Alkaline Phosphatase (AP) or Horseradish Peroxidase (HRP) Polymer permits binding of up to 100 HRP molecules and up to 20 antibody per backbone

Horseradish peroxidase The enzyme horseradish peroxidase (HRP), found in horseradish, is used extensively in molecular biology applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule In the presence of H202 (hydrogen peroxide) DAB (3,3'-Diaminobenzidine) is converted to an insoluble brown reaction product and water by the enzyme HRP DAB + H202 ----------HRP----------> DAB ppt + H20 DAB ppt – insoluble, brown

IHC: New markers for sinusoidal cells in human lymph nodes Martens, Kzhyshkowska et al, J Pathology, 2006

Literature Current protocols in molecular biology www.methods.info www.dako.com

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