Drosophila embryo antibody staining protocol
(for fluorescently tagged 2o and 3o reagents)


Reagents needed:
Fix Block Wash
10x PBS, pH 7.0
(200mM KPO4, 140 mM NaCl)
500 mM EGTA pH 7.4
37% formaldehyde (Sigma)
10x PBS
Triton X-100
BSA
10x PBS
Triton X-100

Alternative fix
0.5 M Pipes pH 6.9
500 mM EGTA pH 7.4
1 M MgSO4
37% formaldehyde (Sigma)

Other
methanol
Drosophila saline (0.7% NaCl; 0.03% Triton X-100)
50% bleach
heptane
FITC, Cy5, and/or LRSC-conjugated 2o and 3o reagents (Jackson)
5x PDA (3 mgs PDA in 1 ml 60% glycerol) (store for up to 2 wks @ -20oC)
Tris pH 8.8

Methods
1) Make a stock soln that is 1.33x PBS and 67 mM EGTA ("in situ fix") OR one that is 100 mM Pipes, 2 mM EGTA, and 1 mM MgSO4 ("PEMFA buffer"). This can be filtered sterilized and stored for ~ 1 yr.

2) Make PT (1x PBS pH 7.0, 0.1% Triton X-100) and PBT (1x PBS pH 7.0, 0.1% Triton X 100, 2% BSA). I make PT in 1 liter volumes, filter sterilize it, and store it at room temp. To make PBT, I put 50 mls of PT in a 50 ml conical and add 1 g of BSA. PBT spoils very quickly; I make up 50 mls at a time and store it at 4oC for 2-3 days.

3) Prepare labelled tubes (eppies, 15 ml Falcons or 50 ml Falcons, depending on the number of embryos to be fixed) with 50% heptane: 50% fixative. Fixative is: 3 parts in situ fix (or 4 parts PEMFA buffer) plus 1 part 37% formaldehyde.

4) Wash staged embryos in Drosophila saline. Rinse with deionized water (diw).

5) Dechorionate in 50% bleach for 2-3 min. Rinse thoroughly with diw.

6) Fix for 20-30 min. with gentle shaking.

7) Replace the fixative with methanol and devitellinize the embryos by shaking. Devitellinized embryos will sink to the bottom of the tube.

8) Aspirate off the heptane and methanol. Rinse the embryos 4-5x with methanol. The embryos can be used immediately or stored at -20oC in MeOH or EtOH for at least one year.

9) Rehydrate embryos in 50% 1x PBS pH 7 (or PT), 50% MeOH, then 100% 1x PBS (or PT).

10) Block embryos in PBT for 1-2 hrs at 4oC.

11) Incubate embryos overnight at 4oC with 1o ABs in PBT. I typically stain ~ 30 ul of packed embryos in 300-600 ul of AB. The incubation can be done with no, or gentle, shaking.

12) Wash embryos 10x over 1 hr with PT.

13) Incubate embryos with 2o antibodies for 2 hrs at 4oC.

14) Wash as before.

15) Make mounting media by diluting 1 part 5x PDA into 5 parts 0.1M Tris-HCl pH 8.8 KEEP STOCK AS WELL AS WORKING MOUNT IN THE DARK AS MUCH AS POSSIBLE.

16) Incubate embryos overnight in mounting media, mount on glass slides and seal with nailpolish.

For optimal imaging, slides should be stored at 4oC in the dark and images collected within 1-2 days.

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