Anti-GFP/BrdU In Situ Immunostaining

Materials
24 well dishes
Millicell 0.4 µm inserts (Millipore PICM01250) - coated with fibronectin
Culture medium
PBS
Fixative:  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 serum
Horse serum
BSA
2N HCl
DAPI Fluorescent mounting medium (Vectashield, Vector Labs H-1200)
Glass microscope slides and cover slips

Procedure

1. Coat Millicell inserts with fibronectin as described by Millipore.
2. Cultivate tissue at 37ºC on fibronectin coated well inserts in a 24-well dish.
3. Remove well inserts and cut out mesh insert with a scalpel, forceps and dissecting microscope
4. 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.
5. Remove solution and wash 3x by adding 1 mL PBS, 3' each.
6. 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.
7. Post-fix in 1 mL 4% paraformaldehyde x15', room temp.  Wash 3x in PBS.
8. Denature DNA with 1 mL of 2 N HCl x30'.  Wash 3x in PBS.
9. Block in 1 mL 10% Horse serum, x30' at room temp.  Wash 1x in PBS.
10. Add 0.3 mL Biotinylated anti-BrdU (1:300, with 1% horse serum in PBS).  Incubate at 4ºC o.n. or 2 hr. at room temp.  Wash 3x in PBS.
11. Dilute 1 µL 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. 
12. Remove antibody and add 1 mL PBS.  O.K. to store at 4ºC.
13. Use a scalpel to remove membrane from well inserts.  Spot 25-50 µL 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 4ºC.