Marco Sassoè

1. Exploring synaptic circuits with antibodies and confocal microscopyPart IPhD School of NeuroscienceTurin UniversityJune 2009 Marco Sassoè

2. Common problems in immunocytochemistry: • Preservation of antigenicity (antigens may be destroyed by chemical fixatives, organic solvents, high temperatures…) • Efficiency of antibody binding to relevant epitopes (may be altered by pH, ionic force) • Background labelling (importance of choice of blocking agent) • Non-specific labelling (importance of appropriate controls)

3. Antibodies • Soluble immunoglobulins: large proteins (150.000 kD) with two binding sites, each recognizing an “epitope” • The epitope may be found in any type of molecule, including proteins (but also in synthetic molecules) • Epitopes present in proteins may be masked by chemical fixatives or, conversely, an epitope that is not present in the native protein configuration may be exposed in the fixed molecule • Most antigens are complex and contain several epitopes, so the immune system responds by producing antibodies to distinct epitopes

4. Antibodies have two light chains and two heavy chains • The term “xenotype” refers to the species producing the antibodies • There are 5 heavy chain isotypes: IgG, IgA, IgM, IgE, IgD • IgG isotypes are further subdivided into the IgG1, IgG2a, IgG2b and IgG3allotypes • Common immunolabelling protocols are based on an indirect labelling scheme (advantages: signal amplification, versatility/economicity; disadvantages: nonspecific binding of secondary antibodies) Secondary antibodies may be specific for the species in which the primary antibody was generated (xenotype), the isotype and the allotype. A careful choice of secondary antibodies may thus help to reduce nonspecific staining considerably

5. Antisera • A preparation containing the immunoglobulins from an animal’s serum • Each antiserum contains a wide array of immunoglobulins, each recognizing distinct epitopes of the antigen used for the immunization • However, in fixed tissue, many if not all of these antigenic sites may be destroyed • In addition, among antisera that stain something in tissues, many may stain spurious antigens (cross-reactivity), and only a few will recognize the original molecule (importance of adsorption controls)

6. Affinity-purified Antisera Most antisera probably have only one or two clones of antibodies that recognize the relevant antigen, the rest of the antibodies being irrelevant (when not producing artifacts!). So, they functionally act as monoclonal antibodies! Affinity purified antisera: they are purified by binding to the appropriate antigen. They are less prone to artifact and tend to give lower background staining

7. Monoclonal antibodies • A single clone of immunoglobulins raised against a specific antigen (each B-cell produces antibodies which specifically bind to a single epitope of the antigen) • Monoclonal antibodies are obtained from an hybridoma, which is derived from the fusion of B-cells with neoplastic B-cells isolated from a myeloma tumor (Kohler and Milstein, 1975) • Hybridoma cells are grown either in culture or by injecting them intraperitoneally in a host animal. In this case, the host animal builds up a fluid (ascites), which contains a high concentration of monoclonal antibodies. In either case, the culture fluids or the ascites containing the antibodies are purified by precipitating the antibodies with protein A • Even the purest monoclonal antibodies provide no assurance that the epitope recognized in tissue sections belongs to the antigen used for the immunization (they require the same type of controls!)

8. Affinity of antibody-antigen interactions • The interactions between antibodies and their antigens are reversible Not all antibodies have the same affinity for their respective ligands: low affinity antibodies bind antigen weakly and tend to dissociate readily, whereas high affinity antibodies bind antigen more tightly and remain bound longer • Factors that influence antibody-antigen interactions are hydrogen bonds, ionic bonds, hydrophobic interactions and van der Waal forces. The strength of these non-covalent interactions is relatively weak (compared to a covalent bond), and largely depends on a very close fit between the antigen and the antibody. This fit requires a high degree of complementarity, which is the basis of the specificity that characterizes antigen-antibody interactions. Nonetheless, antibody-antigen interactions are tight, with affinities in the range of 10-10-10-11 M • Cross-reactivity occurs when two different antigens share an identical epitope, or if antibodies specific for one epitope also bind to an unrelated epitope possessing similar chemical and structural properties

9. Adequate controls for immunocytochemistry • The knock-out test Stain tissue from a wild-type mouse and a KO mouse in which the antigen of interest has been eliminated by transgenic engineering. An even better choice is the use of inducible or spatially-restricted deletion of the protein of interest, which could minimize up or downregulation of other proteins Limitations Applicable only in mice (requires additional controls in other species) The antigen recognized by the antibody may be present not on molecules whose gene was deleted, but on another downstream molecule (in a metabolic or enzymatic pathway) In many KO mice, the original protein is not entirely eliminated, and the portion that remains may have no function, but still stain with the antibody Antibodies bind with low affinity to numerous tissue constituents, and even antibodies that are known to give a specific staining in normal conditions can give rise to non-specific signals in tissue obtained from KO mice

10. Adequate controls for immunocytochemistry Western Blot The antibody should recognize only one antigen in the tissue and this must be of the appropriate molecular weight. Limitations Antibodies that recognize two or more bands may identify the same target molecule in different states of post-translational modification (or in oligomeric forms). In that case, the use of such an antibody requires verification with other antibodies raised against different parts of the same molecule Many antibodies that work well in WB do not recognize the target antigen after it has been distorted by the fixation process

11. Adequate controls for immunocytochemistry • The adsorption test The antibody is pre-adsorbed with a large excess of diluted antigen Limitations The antigen may contain an amino acid sequence that is shared among a group of structurally-related proteins (may require adsorption against structurally related antigens) Not applicable to monoclonal antibodies (they are screened for their binding to the target, and therefore will always pass a preadsorption test, even if they stain something entirely different in tissue). This test is also meaningless for affinity-purified antibodies (for the same reason)

12. Adequate controls for immunocytochemistry • The dilution test The antibody is diluted to the point where it just stains the the sites of interest (can be combined with adsorption tests) • DNA transfection DNA of the target protein (and possibly of protein isoforms) is transfected into cells that normally do not make that protein. However, this control does not prove that the antibody will only stain its target in the tissue • Convergent data a. Compare staining pattern with other, well-characterized antibodies b. Apply a combination of in situ hybridization and immunocytochemistry (not always applicable) c. Compare staining pattern of two or more antibodies raised against distinct epitopes on the same target protein d. If the subcellular distribution of the target molecule is known, the staining pattern should conform to this distribution (not easily applicable to ubiquitous molecules).

13. 1. What immunogen was used to raise the antibody? Complete information on the antigenmust be provided (includingfull sequence) • 2. What is the evidence that the antibody binds specifically to the expected target molecule in the tissue of interest? Western Blot (in the same tissue and species used for IHC) • 3. What controls can be done to insure that the antibody binds in (fixed) tissue only to its target molecule? preadsorption test – knockout test – convergent data (antibodies raised against the same molecule in different species; antibodies raised against different components of the same target molecule)

14. Final notes A single bleed from a rabbit is ~25 ml, half of which consists of serum after the red blood cells have been eliminated. Each bleed is essentially a unique combination of antibody clones. Hence, different batches of a polyclonal antiserum may have entirely different staining properties (it is important to note the lot number of antibodies, i.e. the code number for the animal that produced the antiserum and the bleed). Lack of staining for a molecule of interest cannot be interpreted as absence of that molecule (other possible causes are insufficient affinity of the antibody, suboptimal tissue processing or IHC protocol, epitope masking) Antibodies are made against a globular, aqueous phase of a protein or peptide, and they recognize epitopes that are exposed on the surface of that protein in the acqueous state. However, fixation is based on a chemical reaction that may change the conformation of the molecule and mask the antigenic epitopes. In addition, some epitopes may be masked by other (associated) proteins. In other words, whether an antibody is capable of recognizing its epitope or not critically depends on the method and also on the subcellular location of the protein

15. Preservation of structure and antigenicity • High variety of immunolabelling protocols, due to experimental conditions, antigen properties, antibody reactivity … • For a successful immunostaining of an antigen in a tissue section there must be: • Retention of the subcellular distribution of the antigen (at the same sites that it occupied in the living organism) → fixation • Permeability of tissue to the antibody molecules (cross-linking of proteins by fixation impedes antibody penetration – weak fixation enhances penetration at the expense of structural preservation) • The epitopes must be accessible to the primary antibody (cross-linking due to the fixative is likely to mask epitopes → false negative results)

16. Effects of formaldehyde fixation One of the most common fixatives is a 4% solution of formaldehyde (a ten-fold dilution of formalin). Knowledge of the chemistry of formaldehyde has mainly derived from investigations in the tanning industry, where bovine dermal collagen is converted into leather! The concentration of free formaldehyde in a diluted aqueous solution is very low, as most formaldehyde is present as methylene glycol, which is formed by addition of a molecule of water to one of formaldehyde. It is free formaldehyde that enters the chemical reaction of fixation Formaldehydepenetratesquiterapidly in tissue (5 mm in ~2 h). The chemicalreactionsofformaldehydefixation are slow and itisgenerallyagreedthatgoodstructuralpreservationrequires at least 24 h in formaldehyde. In addition, tissue-boundformaldehyde can beremovedbyprolongedwashing in water. The reactionofformaldehydewithproteinsoccurs in twostages, onefairlyrapid (hours) and the secondmuchslower (days). In the first stage, formaldehydecombineswithproteins, especiallywith the amino groupsoflysine and the nitrogen atomsof peptide linkages. Thesereactions can bereversedbywashing in water.

17. In the second stage, which is slower, the hydroxymethyl groups react with other nitrogen atoms. The resulting cross-links, known as methylene bridges, are stable and account for the rigidity of tissues fixed with formaldehyde. Formaldehyde is more reactive in a basic environment. Therefore, fixation can be improved by perfusing initially with a low pH solution of formaldehyde, to allow a better tissue penetration, and then shifting to a high pH solution that makes the formaldehyde more reactive: Ph-shift protocol • Cross-linkingbyformaldehyde can maskepitopes and impair the penetrationof the antibodies in the tissue. • There are severalwaystoimprove the accessofantibodiestotissue antigens thathavebeenmaskedbyfixation: • Cryofixationfollowedbycryostatsectioning and brief (30 sec) fixation in cold (-20°C) methanoltostabilize the sections • Minimal fixationwithformaldehyde • Antigenretrievalmethods (Aldehydefixationisreversible, henceheatingtissueto 95°C in the presenceofanacidic pH – fovoring the conversionofaldehydestoorganicacids – can reduce the oxydationreactionsthatoccurduringfixation). Thismayrelieve the sterichindrances or specificconfigurationsthatpreventantibodiesfromreaching the epitopes. • Anothermethodisprovidedby the useof a peptidase (trypsin, pepsin) to strip surface peptide sequences off a fixedprotein, whichmay show epitopesthatwerestericallyinaccessible in the fixedprotein (http://publish.uwo.ca/~jkiernan/FixAnti2.pdf)

18. NR2A – no pepsin NR2A – pepsin WT KO WT KO Watanabe et al., Eur. J. Neurosci. 1998 Whether an antibody is capable of recognizing its epitope or not critically depends on the method! This means that the specificity of the immunolabelling must be verified under each experimental condition

19. Giustetto et al., Neuroscience 1997 Sassoè-Pognetto and Ottersen, J. Neurosci. 2000

20. PSD-95 Sassoè-Pognetto et al, J. Comp. Neurol. 2003

21. Panzanelli et al, Eur. J. Neurosci. 2004

22. Whether an antibody is capable of recognizing its epitope or not also depends on the subcellular localization of the antigen. In other words, some epitopes are masked by (associated) proteins in a subcellular compartment-dependent manner The epitope recognized by the N-terminal antibody is masked in conventionally prepared tissue, whereas the epitope at the C-terminus is not. After antigen retrieval (pepsin treatment) the epitope at the N-terminus also becomes accessible(from Lorincz and Nusser, J. Neurosci. 2008)

23. References • Saper CB and Sawchenko PE (2003) Magic peptides, magic antibodies: guidelines for appropriate controls for immunocytochemistry. J. Comp. Neurol. 465:161-163. • Saper CB (2005) An open letter to our readers on the use of antibodies. J. Comp. Neurol. 493:477-478. • Rhodes KJ and Trimmer JS (2006) Antibodies as valuable neuroscience tools versus reagents of mass distraction. J. Neurosci. 26:8017-8020. • Schneider Gasser EM, Straub CJ, Panzanelli P, Weinmann O, Sassoè-Pognetto M, Fritschy JM (2006) Immunofluorescence in brain sections: simultaneous detection of pre- and postsynaptic proteins in identified neurons. Nat. Protocols 1:1887-1897. • Lorincz A and Nusser Z (2008) Specificity of immunoreactions: the importance of testing specificity in each method. J. Neurosci. 28:9083-9086. • Fritschy JM (2008) Is my antibody staining specific? How to deal with pitfalls of immunohistochemistry. Eur. J. Neurosci. 28:2365-2370. • Saper CB (2009) A guide to the perplexed on the specificity of antibodies. J. Histochem. Cytochem. 57:1-5. Illustration credit: Karl Garsha http://www.itg.uiuc.edu/people/garsha/documents/Immunolabeling.pdf#search=%22garsha%20concepts%22