清华大学：现代生命科学实验教学中心《细胞生物学实验》教学课件（讲稿）Immunofluorescence Observation of Microtubule

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