2 Immunofluorescence 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)
3 Preparation 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.
4 epifluorescencebright fieldbright image against dark backgroundcorresponds to location of antigen
6 Counter Staining with Fluorescent Dyes DAPI stains only DNACounter Staining with Fluorescent DyesEtBr stains DNA and RNA
7 Dual-Labeling Experiments determine extent of co-localizationuse 1o antibodies from different species and 2o antibodies labeled with different fluorochromeslabel 1o antibodies with different fluorochromes
8 Immuno-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)
12 Characterizing Antibodies Use same antigen with different antibodiesQuantify by serial dilutionsIFA
13 Conventional 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
14 measure absorbance with ELISA microplate reader
16 Generic 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
17 Biotin-Avidin Detection Systems label 1o- or 2o-Ab with biotindetect with avidin labeled with markerhigh affinity may increase sensitivitymore steps
18 TECHNIQUEGENERAL 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?