Non fluorescent in situ straining of embryos


Hybridization solution: (for 20mL)
            10mL formamide (Sigma)
              5mL SSC 20x (stored at RT)
              2mL Denhardts 50x (Open Biosystems #SPB1140 - stored at 4°C)
            500uL yeast tRNA (Ambion #7188 10mg/mL)
              1mL herring sperm sheared DNA (Open Biosystems #SPB1136 10mg/mL)
            500uL heparin (10mg/mL)
            100uL EDTA 0.5M
            200uL Tween-20 0.1%
            200uL CHAPS 25% (CHAPS 5g Sigma #C3023)
            500uL DEPC water
Store at -20°C. The solution can be used for 1-2 months.





In situ staining of embryos

 Collect eggs
 Dechorionate eggs in bleach 2-4min
 Wash with distilled water
 Fix in 4% FA + heptane 20min
 Remove the formaldehyde solution, add methanol and shake to devitellinize embryos
  wash with methanol 3 times
 Keep at –20°C in methanol if necessary.

In situ staining
 Rehydrate twice in 50%methanol+PBT* (PBT* = PBS, O,1% Tween 20, RNAse free) for 2min
 2 washes PBT* for 2min
 Fix in 4% FA 15min (tubes stored at -20C near the fly room)
 One quick wash PBT*
 5 washes PBT* 5min each
 Incubate in 50% PBT*+HYB for 5min. From this step, do not rotate embryos because they are very fragile when in the HYB solution
 Incubate in HYB for 5 min
 Incubate in HYB for 1h at 55C
 Incubate overnight at 55C with the DIG-labeled RNA probe (4uL in 200uL, denaturate the probe at 80C for 1min prior to the first utilization)
 Wash in HYB for 20min at 55C
 Wash in 50% HYB+PBT for 20min at 55C (PBT = PBS, O,1% Tween 20)
 4 washes in PBT 20min each, at 55C, the last one at RT
 Incubate with anti-DIG-HRP (Roche, 1/500) or anti-DIG-PA (Roche, 1/5000, stored at 4C in the “ABCDE” box) for 2h at RT
 3 washes in PBT 10min each

AP reaction:
 3 washes in AP staining solution 5min each
(the AP staining solution is:     5mL Tris Hcl pH=9.5
                1mL NaCl 5M
                2.5mL MgCl2 1M
                0.5mL Tween 10%
                total=50mL)
 prepare the NBT/BCIP stainining solution: 500uL staining solution + 10uL NBT/BCIP (pink box at 4C) and incubate the embryos in this solution.
 wash the reaction under the microscope. Staining should appear within 5-10min.
 One quick wash in PBT
 3 washes in PBT for 10min
 Mount in 80% glycerol (70uL for a large coverslip)


HRP reaction:
 HRP reaction: use TSA-FITC (NEN, 4uL of TSA-FITC in 200uL of amplification buffer) for 8-10-12min at RT. (decide based on the results of the AP staining)
 One quick wash in PBT
 3 washes in PBT for 10min
 Mount in 80% glycerol (70uL for a large coverslip)