Modern Techniques Základní koncepce užití imunofluorescence na pletivové a na buněčné úrovni

Principy detekce epitopů v buňkách in situ a v izolovaných frakcích

Principy a způsoby detekce proteinů a jiných molekul Imunohisto/cyto-chemie

- kombinuje histologické, imunologické a biochemické techniky k identifikaci specifických tkanivových (histo) a buněčných (cyto) komponent prostřednictvím specifické reakce antigen-protilátka, kde je protilátka značena viditelnou značkou. Vizualizuje se tím na kvalitativní, a případně i na kvantitativní ůrovni distribuce a lokalizace specifických buněčných komponent v buňce a ve tkanivu Základní pojmy:

• Immunohistology • • Immunocytochemistry • Immunofluorescence • Immunoglobulins • Antigens • Haptens • Epitopes • Antibodies

Imuno-histo/cyto-logicke/chemicke metody:

- based on the antigen-antibody reaction

- method that visualize distribution and localization of specific antigen or cellular components in separated tissues, tissue sections, cells, or isolates. Compared to other bio-techniques that are based on the antigen-antibody reaction such as , or western-blot, immuno-histo/cyto-chemistry provides in situ information. Základní pojmy:

Immunohistochemistry (IHC) Detection of a target in tissue using an antibody and subsequent visualization of the antigen- antibody complex using a chemical reaction to produce a color change

Immunocytochemistry (ICC) Detection of a target in cells using an antibody and subsequent visualization of the antigen- antibody complex using a chemical reaction to produce a color change

Immunofluorescence (IF) Detection of a target in cells or tissues using an antibody and subsequent visualization of the antigen-antibody complex using a fluorophore

Methods use labeled antibody to trace antigen-antibody interactions and the antibody can be detected by several methods.

Antibody conjugated to: - enzyme (horseradish peroxidase or alkaline phosphatase) for color detection - fluorophore for immunofluorescence - particles Základní pojmy:

Immunohistochemistry (IHC)

- refers to the process of detecting antigens (e.g., proteins) in cells of a tissue by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Základní pojmy:

Immunohistochemistry (IHC)

- refers to the process of detecting antigens (e.g., proteins) in cells of a tissue by exploiting the principle of antibodies binding specifically to antigens in biological tissues.

Immunohistochemistry immunohistofluorescence Základní pojmy:

Immunocytochemistry (ICC) - used to anatomically visualize the localization of a specific protein or antigen in cells by use of a specific primary antibody that binds to it.

- ICC allows researchers to evaluate whether or not cells in a particular sample express the antigen in question.

- In cases where an immunopositive signal is found, ICC also allows researchers to determine which sub-cellular compartments are expressing the antigen.

Immunocytochemistry immunocytofluorescence Základní pojmy:

Immuno – refers to immunological technique (ie. the binding of antibodies to an antigen) Histo – refers to tissue (cells with the extracellular matrix) Cyto – refers to cells (cells without the extracellular matrix) Chemistry – chemical method of detection (ie. change in color) Fluorescence – detection by emission of light from an excited fluorophore

Tissue (-histo) Cells (-cyto)

Chemical detection Immunohistochemistry Immunocytochemistry (-chemistry) Fluorescent detection Immunohistofluorescence Immunocytofluorescence (-fluorescence) Základní pojmy:

Antigen

- a substance which when introduced into a body evokes immune response to produce a specific antibody with which it reacts in an specific manner

- Complete antigen - Incomplette antigen (haptens)

Complete antigens are able to induce antibody formation and can react specifically with them

Haptens are unable to induce antibody formation but can become immunogenic (capable of inducing antibodies) when covalently linked to carrier proteins.

Epitope The smallest unit of antigenicity Epitops (antigen determinants) – particular chemical groups on a molecule that are antigenic

Antibody - A special group of soluble proteins that are produced in response to foreign antigens Protilátky (antibodies)

Imuno-cyto/histochemie: hlavní výhody

• Protilátky se vážou na antigen specificky • umožňuje lokalizovat jenom žádané proteiny • umožňuje identifikovat buněčné procesy (metabolizmus, dělení, apoptóze…) • Poskytuje prostorovou lokalizaci

Základní pracovní postup metody Základní pracovní postup metody Základní pracovní postup metody Základní pracovní postup metody Základní pracovní postup metody

HRP – Horse Radish Peroxidase Základní pracovní postup metody Immunofluorescence (IF) - Applications include the evaluation of cells in suspension, cultured cells, tissue, beads and microarrays for the detection of specific proteins. - IF techniques can be used on both fresh and fixed samples. - In IF techniques, antibodies are chemically conjugated to fluorescent dyes such as fluorescein isothiocyanate (FITC) or tetramethyl rhodamine isothiocyanate (TRITC). - These labeled antibodies bind (directly or indirectly) to the antigen of interest which allows for antigen detection through fluorescence techniques.

Advantages of direct immunofluorescence - shorter sample staining times and simpler dual and triple labeling procedures. - In cases where one has multiple antibodies raised in the same species, for example two mouse monoclonals, a direct labeling may be necessary.

Disadvantages of direct immunofluorescence - Lower signal, generally higher cost, less flexibility and difficulties with the labeling procedure when commercially labeled direct conjugates are unavailable. Advantages of indirect immunofluorescence - different sensitivity than direct immunofluorescence. - there is amplification of the signal in indirect immunofluorescence because more than one secondary antibody can attach to each primary antibody. - commercially produced secondary antibodies are relatively inexpensive, available in an array of colors, and quality controlled.

Disadvantages of indirect immunofluorescence - The potential for cross-reactivity and the need to find primary antibodies that are not raised in the same species or of different isotypes when performing multiple-labeling experiments. - Samples with endogenous immunoglobulin may exhibit a high background. Důležité vlastnosti protilátek, které ovlivní výsledek

• monoklonální nebo polyklonální • indukována vůči celé molekule, N- terminálnímu konci, C-terminálnímu konci, nebo specifickým aminokyselinám • ascites, supernatant, serum monoclonal versus polyclonal antibodies • Monoclonal • Polyclonal – Mouse or rabbit – Many different species hybridoma – Tends to have more non- – Tends to be ‘cleaner’ specific reactivity – Very consistent batch- – Can have very different to-batch avidity/affinity batch-to- – More likely to get false batch negative results – More likely to have success in an unknown application Make sure your antibody is validated for your application!!!

Důležité předpoklady k přípravě imunofluorescence • selekce protilátek • kontroly • příprava a fixace • přímá metoda detekce materiálu • nepřímá metoda detekce • řezání • Imunoenzymatická • „antigen Retrieval“ detekce • blokace • fluorescenční detekce • multiple labeling Je nutné na tohle myslet předem, protože to může mít vliv na přípravu vzorků ! Fixation:

Tissue preparation is very important for immunohistochemistry. To ensure the preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential. However, inappropriate or prolonged fixation may significantly diminish the antibody binding capability.

Sectioning: Parafin sections Paraffin wax has remained the most widely used embedding medium in routine histological laboratories. Most of material for immunohistochemistry is formalin-fixed, paraffin-embedded. Paraffin sections produce satisfactory results for the demonstration of majority of tissue antigens Frozen sections Certain cell antigens do not survive routine fixation and paraffin embedding. Disadvantage of frozen sections includes poor morphology, poor resolution at higher magnifications and special storage needed

Whole Mount Preparation: Small blocks of tissue (less than 5 mm thick) can be processed as whole mounts. The advantage of whole mount preparations is that the results provide three dimensional information about the location of antigens without the need for reconstruction from sections. However, the major limitation of using whole mounts is antibody penetration that may not be complete in the tissue, resulting in uneven staining or false negative staining. Sectioning

• Paraffin • Frozen – Must be heated and – Better survival of many processed through antigens xylenes and alcohols – – Poor morphology ruins some antigens – Poor resolution at higher – Most commonly used magnifications – BEST if not stored more – Special storage than two weeks – lose – Cutting difficulty antigenicity after that time

Improving antibody penetration

. Need this for intracellular (cytoplasmic, nuclear) or membrane components when epitope is inside cell membrane . Detergents most popular  Triton-X  Tween  Also decreases surface tension – better coverage . Can’t use for membrane proteins . Acetone/Methanol  Precipitate proteins outside cell membranes- more accessible . Saponin  Punches holes in cell membrane – holes close up when removed

Effects of fixation on small peptides and cell membrane antigen stability. Formalin fixation stabilizes small peptides and cell membrane antigens, preventing leakage or diffusion, even after sectioning. With acetone fixation, small peptides leak through the permeabilized cell membrane. Formalin fixation stabilizes membrane antigens in cryostat sections, but small peptides leak away. Blocking:

Background staining may be specific or non-specific. Inadequate or delayed fixation may give rise to false positive results due to the passive uptake of serum protein and diffusion of the antigen. Such false positives are common in the center of large tissue blocks or throughout tissues in which fixation was delayed.

The main cause of non-specific background staining is non-immunological binding of the specific immune sera by hydrophobic and electrostatic forces to certain sites within tissue sections. This form of background staining is usually uniform and can be reduced by blocking those sites with normal serum.

Autofluorescence or natural fluorescence exists in some tissues and can cause background problems when fluorescent dyes are used in the experiments. The simplest test is to view the tissue sections with a before any antibody incubation. If autofluorescence is detected in the tissue sections, the best solution is to avoid use of fluorescent method but choose enzyme or other labeling methods. Blocking

Background staining: • Specific – Polyclonal antibodies – impure antigen used – Inadequate fixation – diffusion of antigen – often worse in center of large block • Non-specific – Non-immunologic binding – usually uniform – Endogenous peroxidases – Endogenous biotin Blocking Controls:

Special controls must be run in order to test the protocol and for the specificity of the antibody being used.

Positive control is to test a protocol or procedure and make sure it works. It will be ideal to use the tissue of known positive as a control. If the positive control tissue showed negative staining, the protocol or procedure needs to be checked until a good positive staining is obtained.

Negative control is to test for the specificity of an antibody involved. First, no staining must be shown when omitting primary antibody or replacing an specific primary antibody with normal serum (must be the same species as primary antibody). This control is easy to achieve and can be used routinely in immunohistochemical staining.

Second, the staining must be inhibited by adsorption of a primary antibody with the purified antigen prior to its use, but not by adsorption with other related or unrelated antigens. This type of negative control is ideal and necessary in the characterization and evaluation of new antibodies but it is sometimes difficult to obtain the purified antigen, therefore it is rarely used routinely in immunohistochemical staining. Controls

• Positive control – Best is tissue with known specificity • Negative control – Best is IgG from same species immunized against non-biologic molecule – Can also use non-immunized serum from same species – Pre-absorption with the purified antigen Multiple Labeling

• - It is often useful to be able to stain for two or more antigens in one common tissue section. • - this can be achieved by immunofluorescence method using different fluorescent dyes. • - multiple staining can also be done with peroxidase conjugated antibodies developed with different chromogen substrates to produce the end products of different colors. • - there are three basic approaches in planning multiple staining: parallel, sequential and adjacent. • - the antibody dilution and condition are also important factors to be considered. • - appropriate color combination is also crucial since improper color combination may produce poor result and fail to demonstrate multiple antigens in the same section. • - for best result, the careful design and test of multiple staining protocols are necessary. Multiple Immunofluorescence

GAMMA-AMINOBUTYRIC ACID

Triangles on surface of ultrathin section (hatched) represent the antigen against which the primary antibody (A) was raised. The secondary antibody (B) is attached to a colloidal gold particle (Au); in this case, a gold particle with a radius (r) of 7.5 nm. With an effective IgG diameter of 8 nm, the maximum projected distance between epitope and gold particle will be in the range of 23 nm (two IgG diameters plus gold particle radius). This is in good agreement with experimental data (Fig. 2). With a secondary Fab (diameter ~4 nm) in lieu of a secondary IgG (diameter ~8 nm), the length of the antibody bridge will be reduced correspondingly.

Quantum dots selectively label endoplasmic reticulum Základní imunometody detekce proteinů

• ELISA • Gel Electrophoresis • • Immunoprecipitation • Enzyme assays • Immunohistochemistry ELISA (Enzyme Linked ImmunoSorbent Assay) ELISA (Enzyme Linked ImmunoSorbent Assay) ELISA (Enzyme Linked ImmunoSorbent Assay)

Direct ELISA. Indirect ELISA.

Sandwich ELISA. ELISA (Enzyme Linked ImmunoSorbent Assay) Types of ELISA Methods: Direct ELISA: Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or quantify antigen concentration. The specificity of the primary antibody is very important. PROS: minimum procedure; avoids cross-reactivity from secondary antibody. CONS: requires labeling of all primary antibodies - high cost; not every antibody is suitable for labeling.

Indirect ELISA: Primary antibodies are not labeled, but detected instead with enzyme-conjugated secondary antibodies that recognize the primary antibodies. PROS: secondary antibodies are capable of signal amplification; many available secondary antibodies can be used for different assays; unlabeled primary antibodies retain maximum immunoreactivity. CONS: cross-reactivity may occur.

Sandwich ELISA: The antigen to be measured is bound between a layer of capture antibodies and a layer of detection antibodies. The two antibodies must be very critically chosen to prevent cross-reactivity or competition of binding sites. PROS: sensitive, high specificity, antigen does not need to be purified prior to use. CONS: antigens must contain at least two antibody binding sites.

Competitive ELISA: The antigen of interest from the sample and purified immobilized antigen compete for binding to the capture antibody. A decrease in signal when compared to assay wells with purified antigen alone indicates the presence of antigens in the sample. PROS: crude or impure samples may be used, high reproducibility. CONS: lower overall sensitivity and specificity.

A) Schematic representation of the antibody array based assay set-up. Protein-protein interactions are detected by capturing target protein, A, with one specific antibody and detecting the interacting partner, B, with another specific antibody. In this assay configuration multiple capture antibodies are printed on each pad and a single detection antibody is applied per pad. This format is amenable to a highly parallel assay and both direct and indirect protein- protein interactions can be detected using this assay. B) A photo of an eight-pad nitrocellulose- coated glass slide on which the capture antibodies are printed. Digital microfluidic Immunocytochemistry in Single Cells Digital microfluidic Immunocytochemistry in Single Cells