2 Immunostaining Not a one size fits all approach General guidelines that can be modified depending on tissue, antigen and antibodies available.Optimization and luck are typically required.
3 Immunostaining General Steps Overview FixRinse (3x, 10 min)Cryoprotect (if using frozen sections, 30 – 60% sucrose O/N, 4°C)Imbed (paraffin, frozen, gelatin)SectionRinse (3x, 5 min)PermeabilizeNon-specific block (1 hr, RT)Primary Antibody (1 hr -72 hr, 37 °C, RT 4°C)Secondary Antibody (1 hr, RT or 37°C)Mounting media and coverslip
4 Fixatives Options Formaldehyde (CH2O) Formalin, 10% (saturated, 37% formaldehyde) ***Often has MeOHParaformaldehyde (4% PF); polymerized formaldehydeGlutaraldehyde (high autofluorescence, ideal for EM)Joy Juice (MeOH, acetone causes denaturation)Time is important.Fixatives cause crosslinking between proteins, leaving samples in fixative more than hrs can lead crosslinking “blockade” of antigen.Some antigen-antibody interactions are “fixative sensitive”, so read antibody insert thoroughly.
5 To infuse or not to infuse When should you infuse?Highly vascularLargeMultiple organ collectionApproachHeparinize firstAnesthetize till respiration ceasesOpen chestSnip liverInfuse with ml PBS/saline, until fluid running out of liver is clear (nose, paws, internal organs should blanch) via left ventricle.Infuse with 10 – 60 ml fixative. Should get muscle contraction and animal should get stiff like a board.Excise samples and post-fix in fixative for hours….Longer is not better.
6 PermeabilizationGoal: allow antibodies access to intracellular compartmentsDetergents (TX-100, NP-40, Tween-20, CHAPS, Saponin)Epitope dependent…don’t use if antigen is “solubilized” by the detergent.Solvents – minimize lipid barrier, DMSOFixative (PF, acetone, works with cultured cells)Optimization of time, duration and concentration
7 Sectioning Frozen Paraffin Gelatin Advantages: quick, relatively easy, flexibility: works with most antigensDisadvantages: can get cryo-damage, doesn’t preserve morphology as well as paraffinCryoprotect with 30% sucrose (PBS) until sample sinks, usually O/N. Put in 60% sucrose O/N if it doesn’t sink.Section thickness will vary with tissue …10 – 20 µm is goo general rule.Make sure chamber is cold (-20), blade is sharp and clean (wipe with EtOH if new to remove oils)Getting good sections is an art and takes practice and constant optimization/adjustments.ParaffinAdvantages: preserves structure better, tissue samples keep longerDisadvantages: usually requires “unmasking” techniques, requires more preparation time, more likely to be incompatible with antigen-antibody interaction.GelatinUse with vibratomeCan be desirable with brain sections
8 Blocking SolutionPurpose: Non-specific blocking of antigen sites that may share some weak similarity to targe antigen. Prevents antibody from binding off-target sites.Choices:commercial blocking reagentsNF milkserum, ~5%Never use serum from primary Ab host!!Use serum from secondary Ab host, ~5% in PBSDon’t mix goat and sheep antibodies in an experiment!If using a sheep primary Ab, don’t use goat serum to block
9 Primary AntibodyGetting a good primary Ab can be challenging. Trial and Error.PolycolonalAb has multiple antigenic targetsRabbit, guinea pig, goat, sheepNon-renewable. Generating new ab means injecting new animals and getting new serum which means the new antibody will not be identical to the original.Commercial PC Abs will vary slightly from lot to lot, not usually a problem from highly expressed antigens.Formats: serum, IgG purified, antigen purified (ideal)Monoclonal: Ab has one targetmouse, some rabbit monoclonals availableRenewableFormat: ascites, purified
10 Multiple labeling Ideal: Multiple antibodies, each from different speciesi.e. mouse, rabbit, sheepIf no other options, can use two antibodies from the same host, but do “species conversion” where you convert one mouse into a rabbit or one rabbit into a mouse.
11 Secondary AntibodiesWith fluorescence imaging, will need to use secondary Ab’s that are conjugated to fluorophores that are compatible with imaging device.Confocal488 (Cy2, Alexa 488)561 (Alexa 555, Cy3)633 (Alexa 633)If using multiple labeling, make sure your secondary Abs have been “pre-absorbed” against the other primary antibody hosts or you will get cross-labeling.Store in dark.For MP, can use dyes excited at visible wavelengths, typically use 2x the visible wavelength to excite with MP.
12 Negative Controls Negative Controls No primary, no secondary Ab (autofluorescence)No primary Ab, but include secondary Ab (secondary Ab non-specific labeling)Always run one a no primary with every experiment!!!Antigen blockade (typically meaningless if Ab has been affinity purified)Heterologous cell line expressing exogenous target (trasfected cell line).siRNA knock-downIDEAL proof of Ab specificity: Knock-out/null animal.
13 Positive Controls Does Ab recognize target? Heterologous cell line expressing exogenous target (trasfected cell line).Does it label the type of cell I’m interested inVSMC- alpha smooth muscle actin, desminEndothelial cells – PECAM, CD31Muscle fibers – myosin chain isoformsNeurons – there are not a lot of good pan-neuronal Abs, most are specific for specific populations; PG9.5, neurofilament (heavy, intermediate, light)Schwann cells – S100Google it, go to literature
14 Signal low? Try higher primary Ab concentration up to 1:25. Try longer primary Ab incubation up to 72 hr.Avoid fluorophore labeled primary antibodies (lose your signal amplification with secondary Ab.Signal Amplification with Tyramide conjugated fluorophore.HRP conjugated secondary Ab, treat with H2O2, then add tyramide conjugated fluorophore…get higher signal, but some reduced resolution.Look for signal directly at target, don’t be swayed by “higher signal” in other tissues, different cell types have different levels of background signal.
15 Other thoughtsMake your own antibody for commonly used sensitive targets. It’s expensive upfront, but you’ll have the same Ab for many years and you’ll save time repeatedly optimizing new Ab lots.Write the receipt date on commercial Abs. They don’t last forever, even if stored at -20.Aliquot Abs into single use aliquots that are to stored at Multiple freeze thaws are bad.Save all antibody information sheets and generate a catalog, write notes re: titer and sensitivity.Always read the antibody information sheet front to back.Read thru catalogs, they usually have sections of fundamental information.Always, always, always, run a negative control to account for “background” AF.Don’t loose the tree thru the forest. Don’t expect your target to be the highest fluorescing object in the tissue section. Background fluorescence in different tissue varies and may be higher than in your expected target. Compare apples to apples. This is why negative controls are important.Just because an antibody works for western blotting, doesn’t mean it will work for immunolabeling or vice versa.Species specificity. Even though your antibody is supposed to be targeted to mouse protein X, doesn’t guarantee it will or will not recognize that same target in another species.