2Immunofluorescence Used to: General Procedure: localize antigens to specific cells or subcellular structuresdetect and quantify antibodyGeneral Procedure:incubate cells or tissue with antibodydetect Ag-Ab complex with conjugated secondary antibodyfluorescence (examine under UV illumination)enzyme (substrate forms precipitate)
3Preparation of Cells IFA Protocol section and mount tissues grow adherent cells on micro-scope slides or cover slipsaffix suspension cells on microscope slidescarry out incubations in suspension± fixationorganic solventsparaformaldehyde<0.1% glutaraldehyde± permeabilization (eg., detergents)surface labeling of unfixed cellsIFA Protocol1. Prepare cells or tissue.2. Incubate 1o antibody.3. Wash.4. Incubate 2o antibody.5. Wash.6. View under UV illumination.
4epifluorescencebright fieldbright image against dark backgroundcorresponds to location of antigen
6Counter Staining with Fluorescent Dyes DAPI stains only DNACounter Staining with Fluorescent DyesEtBr stains DNA and RNA
7Dual-Labeling Experiments determine extent of co-localizationuse 1o antibodies from different species and 2o antibodies labeled with different fluorochromeslabel 1o antibodies with different fluorochromes
8Immuno-Electron Microscopy prepare samplesoptimize fixation conditionsuse resins that polymerize at RT‘float’ grids on drops (1o and 2o abs, washes)surface of section accessible to antibodies (± 'etching')2o-Ab conjugated with colloidal goldsize ranges from 5-15 nmenzyme linked (electron dense precipitate)
12Characterizing Antibodies Use same antigen with different antibodiesQuantify by serial dilutionsIFA
13Conventional ELISA bind antigen to 96-well microplate (or membrane) neg. (and pos.) controlspurity?incubate with 1o and 2o antibodiesuse soluble chromogenic substrates in 96-well platesquantify Ab or Ag
16Generic Immunoassay Procedure form antibody-antigen complex detect Ag-Ab complexlabeled anti-antibody (2o Ab)labeled protein A or Gdirectly label 1o AbDirectvs.Indirectless stepsless bkg (?)dual labelconvenienceamplification (?)FluorochromesfluoresceinrhodamineEnzyme CrosslinkingAPHRPRadiolabelingiodinationmetabolically (mAbs)Biotinylation
17Biotin-Avidin Detection Systems label 1o- or 2o-Ab with biotindetect with avidin labeled with markerhigh affinity may increase sensitivitymore steps
18TECHNIQUEGENERAL PROCEDURETYPICAL APPLICATIONELISAadsorb Ag to solid supportincubate with Abdetect bound Abquantify Abquantify Agprocess large # of samplesBLOTTINGSDS-PAGE and transferidentify subunit MWquantify Ab?IFAfix cells on slidesubcellular localizationquantify Ag?