in situ+antibody staining 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 + methanol 20min
 Remove the formaldehyde solution and 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, DEPC water)
 2 washes PBT for 2min
 Fix in 4% FA 15min (200mL 37% FA+ 1600mL PBT)
 One quick wash PBT
 5 washes PBT 5min each
 Incubate in 50% PBT*+HYB for 5min
 Incubate in HYB for 5 min
 Incubate in HYB for 1h at 55C
 Incubate overnight at 55C with the DIG-labeled RNA probe (1uL in 300uL, 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
 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, 1/2000) for 2h at RT
 3 washes in PBT 10min each
 HRP reaction: use TSA-FITC (NEN, 4uL of TSA-FITC in 200uL of amplification buffer) for 10min at RT.
AP reaction: use NBT/BCIP
 One quick wash in PBT
 3 washes in PBT for 10min
 H2O2 treatment (if HRP is used for antibody staining): incubate with H2O2 0.3% for 30min
 One quick wash in PBT
 5 washes in PBT for 5min
 Incubate with mouse anti-Cut (DSHB, 1/1000), rabbit anti-Bgal (Cappel, 1/2000)

Antibody staining
 3 washes in PBT 20min each
 Incubate with sencondary antibodies (anti-mouse-biotin(anti-mouse-biotin, 1/1000, plus other secondary antibodies) for 1h
 3 washes 20min each in PBT. Meanwhile, 30min before the next step, prepare the streptavidine-HRP solution by mixing solutions A+B at 1/100 dilution each in PBT (Vectastain, kit ABC)
 Incubate with streptavidine-HRP for 1h (500uL in each tube)
 3 washes 10min each in PBT. Remove as much PBT as you can after the last wash
 Incubate with the TSA-biotin solution (NEN, 4uL TSA-biotin in 200uL of amplification buffer) for the HRP reaction. Usually for 10min (try also 8-14min to get various staining intensities). Tubes should also be rotating during this step.
 Quick wash in PBT
 3 washes 10min each in PBT
 Incubate with streptavidine-Alexa568 or streptavidine-Alexa488 (Molecular Probes, 1/1000) for 30min
 3 washes 10min each in PBT

Mounting
 Remove as much PBT as you can
 Put 55uL of Vectashield mounting medium in the tube and aspirate the embryos as they come up with a P200 pipette tip that has been cut at its tip.
 put the liquid on a slide, cover with coverslip