Indirect Immunofluorescence

Reagents:  NGS-Normal Goat Serum  PBST-0.5% Tween-20/CMF-PBS  CMF-PBS  1°Ab-mouse MF20 (anti-myosin monoclonal)  1°Ab-rabbit myoD (anti-myoD polyclonal)  2°Ab-goat anti-mouse IgG-FITC  2°Ab-goat anti-rabbit IgG-FITC

Procedure:

You will be staining one of your transfected chamberslides with anti-myoD.

You will be staining the other transfected chamberslide and the untransfected one with anti-myosin.

ALL STEPS ARE PERFORMED AT ROOM TEMPERATURE

1.  Block 30 minutes in 1% NGS/CMF-PBS--use 500 ul/well.

2.  Dilute the primary antibodies in 1% NGS/CMF-PBS.  Make 4 mls MF20 diluted 1:5  Make 1.5 mls myoD diluted 1:100

3.  Bind the primary antibodies 1.5 hours at room temperature in 1% NGS/CMF-PBS--use 300 ul/well.

4.  Wash 3X with PBST, 5 minutes each wash-use ~1 ml/well.

THE REST OF THE STEPS ARE PERFORMED IN THE DARK-EITHER PLACE THE SLIDE IN A DRAWER OR COVER IT WITH FOIL

5.  Dilute secondary antibodies in 1% NGS/CMF-PBS.  Make 4 mls anti-mouse FTIC diluted 1:150  Make 1.5 mls anti-rabbit FITC diluted 1:200

6.  Bind secondary antibodies 45 minutes--use 300 ul/well.

7.  Wash 3X with PBST, 5 minutes each wash-use ~1 ml/well.

8.  Wash 1X with CMF-PBS, 5 minutes-use ~1 ml/well.

9.  Mount with 20 ul of Vectashield with DAPI/well, add coverslip.  Drain excess Vectashield by holding slide on its side on a piece of filter paper.

10.  Store at 4°C in a slide box or view immediately.

上一篇：免疫荧光操作（共聚焦显微镜）-Immunofluorescence Protocol for Confocal Microscopy   下一篇：酵母免疫荧光染色--Immunofluorescent Staining of Yeast:SDS Permeabilization Metho