Materials24 well dishesMillicell 0.4 m inserts (Millipore PICM01250) - coated with fibronectinCulture mediumPBSFixative: 4% paraformaldehyde in PBS (pH 7.6)Rabbit anti-GFP (Molecular Probes, A-11122)FITC-goat anti-rabbit IgG (Jackson ImmunoResearch, 111-096-006)Biotinylated anti-BrdU (AbCam, ab2284-125)Streptavidin-Alexa Flour 555 (Invitrogen/Molecular Probes, S21381)Goat serumHorse serumBSA2N HClDAPI Fluorescent mounting medium (Vectashield, Vector Labs H-1200)Glass microscope slides and cover slipsProcedureCoat Millicell inserts with fibronectin as described by Millipore.Cultivate tissue at 37 C on fibronectin coated well inserts in a 24-well dish. Remove well inserts and cut out mesh insert with a scalpel, forceps and dissecting microscope Place inserts in a 2.0 mL Eppendorf tube with 1 mL of blocking agent (10% goat serum, 1% BSA in PBS). Gently flick tube to mix. Remove solution and wash 3x by adding 1 mL PBS, 3\' each. Add 0.3 mL rabbit anti-GFP (1:200 v/v, plus 1% goat serum in PBS) and incubate 1 hr. at room temp. Wash 3x in PBS. Post-fix in 1 mL 4% paraformaldehyde x15\', room temp. Wash 3x in PBS. Denature DNA with 1 mL of 2 N HCl x30\'. Wash 3x in PBS. Block in 1 mL 10% Horse serum, x30\' at room temp. Wash 1x in PBS. Add 0.3 mL Biotinylated anti-BrdU (1:300, with 1% horse serum in PBS). Incubate at 4 C o.n. or 2 hr. at room temp. Wash 3x in PBS. Dilute 1 L fresh streptavidin-Alexa Flour 555 in 1 mL PBS (1:1,000). Spin the dilute antibody at high speed for 30\' to remove particulates. Add supernatant to specimen and incubate 30\' at room temp. Remove antibody and add 1 mL PBS. O.K. to store at 4 C.Use a scalpel to remove membrane from well inserts. Spot 25-50 L of DAPI mounting medium onto a glass slide. Place 1-2 membranes on the DAPI medium. Use a dissecting microscope to be sure to place the tissue side up. Cover with a 24-40 mm glass slip. Paint the edges with clear nail polish. View with immunoflourescence. Store in the dark at 4 C.联系人：蚂蚁淘编辑部Email：service@bioon.com电话：021-54485309传真：021-54485087