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

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

3. 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

4. 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

5. 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

6. 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)

7. 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

8. 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 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

9. 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

10. 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

11. Experimental design for IHC1 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

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

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

14. 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

15. 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+

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

17. 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

18. 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

19. 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).

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

21. 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

22. 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

23. 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

24. 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 + H HRP > DAB ppt + H20
DAB ppt – insoluble, brown

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

26. Literature Current protocols in molecular biology www.methods.info

27. Questions?