Immunofluorescence ObservationofMicrotubule
Immunofluorescence Observation of Microtubule
Cytoskeleton1879, W.Flemming1963, Slautterback. cnidoblast (hydroid)1974, E. Lazarides & K. WeberMF、MT、IFMT:α-tubulin、β-tubulin
Cytoskeleton 1879, W. Flemming 1963, Slautterback. cnidoblast（hydroid） 1974, E. Lazarides & K. Weber MF、MT、IF MT:α-tubulin、β-tubulin
Immunofluorescence Techniuedirect methodindirect methodsecondary antibodyprimary antibodymarkerantigenFig.1.Indirect immunofluorescence technique
Immunofluorescence Technique direct method indirect method Fig.1. Indirect immunofluorescence technique
ReagentPBS4%paraformalclehyde-PBS0.5%TritonX-100-PBS10%NGS-PBSPBST:PBScontaining0.1%Tween-20primaryantibody:mouseantia-tubulin（(200μg/mL)（(1:100PBS)secondary antibody:FITC/TRITC-Conjugated Goat Anti-MouseIgG(1.5mg/mL）（(1:200PBS)NGS:normal goat serumFITC:fluoresceinisothiocyanateTRITC:tetramethylrhodamineisothiocyanate
Reagent PBS 4% paraformalclehyde - PBS 0.5%TritonX-100-PBS 10%NGS - PBS PBST: PBS containing 0.1%Tween-20 primary antibody: mouse anti α-tubulin （200μg / mL）（1:100PBS） secondary antibody: FITC/TRITC-Conjugated Goat Anti-Mouse IgG （1.5mg/mL） （1:200PBS） NGS: normal goat serum FITC: fluorescein isothiocyanate TRITC: tetramethyl rhodamine isothiocyanate
Immunostaining (1)PreparationPlatecells ontocoverslipsRinse cells with PBSFix infreshprepared 4%paraformalclehyde-PBS(50OuL)inPBS for 15minRinse in 3X (5-10) min PBS at RTPermeabilizein0.5%TritonX-100inPBSfor15minRinse 3X (5-10) mineach inPBS
Immunostaining（1） Preparation Plate cells onto coverslips Rinse cells with PBS Fix in fresh prepared 4% paraformalclehyde - PBS(500µL) in PBS for 15min Rinse in 3×（5-10）min PBS at RT Permeabilize in 0.5% TritonX-100 in PBS for 15min Rinse 3× （5-10） min each in PBS
Immunostaining (2)Incubate cells in 10%NGS-PBS solution at 37C for 30 minIncubate with primary antiboday (80uL)at 37℃ for 30-60min/ 4℃ overnightRinseinPBSTatRTfor3X10mineachIncubate with secondary antibody (80uL) at 37C for 30minRinse3X10min inPBSTExaminebyfluorescencemicroscopy
Immunostaining（2） Incubate cells in 10%NGS-PBS solution at 37℃ for 30 min Incubate with primary antiboday (80µL) at 37℃ for 30-60 min / 4℃ overnight Rinse in PBST at RT for 3×10 min each Incubate with secondary antibody (80µL) at 37℃ for 30 min Rinse 3×10 min in PBST Examine by fluorescence microscopy
(1)Results
Results（1）
(2)Results100100um
Results（2）
CostainingDAPIIncubate with DAPI(8OuL) at RT for 5 minRinse 3X 10 min in PBS
Costaining DAPI Incubate with DAPI(80µL) at RT for 5 min Rinse 3×10 min in PBS
Results
Results