Procedure
Stock Solution:
PEM Solution:
125 mM Pipes (pH 7.0)
2.5 mM EGTA (pH 8.0)
1.25 mM MgSO4
Fix Solution:
4% paraformaldehyde (PFA) in PEM (freshely prepared)
PFA = 100 mg into 500 ul H2O + 1ul 1N NaOH, incubate ~70°C (heating block) for ~10min = 20%
200ul PFA + 800 ul PEM = 4%PFA
TBS-TX:
20 mM Tris-HCl (pH 7.5)
130 mM NaCl
1 mM EDTA
0.1% Triton X-100
TBS-TB:
TBS-TX + 0.2% BSA
Dissect Flies in PBS (~300 ul) within 30min
Fix with 250 ul of 4% PFA in PEM for 30 min
Wash with 300 ul of PEM for 10 min x2 times
Incubate with 300 ul of PEM containing 1% NP40 for 30 min
Wash with 300 ul of TBS-TB for 5 min x2 times
Blocking with 250 ul of TBS-TB containing 5% goat serum for overnight at 4°C or 2hrs at room temperature (RT)
Incubate with 250 ul of primary antibody in TBS-TB for overnight at 4°C or 2hrs at RT
Wash with 300 ul of TBS-TB for 10min 3x times
Incubate with 250 ul of secondary antibody in TBS-TB for overnight at 4°C or 2hrs at RT
Wash with 300 ul of TBS-TX for 10 min x3 times
Rinse with 300 ul of PBS for 5 min x2 times
mount on the slide glass with mounting medium containing DAPI