in situ staining of embryos or larval tissues
(protocol used in Chantal Dauphin-Villemant's lab, 2008)

Hybridization solution: (for 20mL)

            10mL formamide (bottle in the cupboard)
              5mL SSC 20x (Invitrogen box above the Northern bench)
              1mL Denhardts 100x (stored at -20°C, transparent plastic box, top drawer, C4)
            500uL yeast tRNA (10mg/mL, stored at -20°C, transparent plastic box, top drawer, C4)
              200uL salmon testis DNA (10mg/mL) - better to put 1mL
            100uL heparin (50mg/mL, diluted in SSC 4x, stored at -20°C, transparent plastic box, top drawer, C4)
            100uL EDTA 0.5M pH=8 (bottle "Virginie" above the Northern bench)
            200uL Tween 10%
            200uL CHAPS 25% (stored at -20°C, transparent plastic box, top drawer, C4)
            2.7mL DEPC water
Store at -20°C. The solution can be used for 1-2 months.


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


Fixation of larval tissues
Clean up dissecting tools with RNase away solution
Dissect larval tissues in PBT* = PBT made from DEPC water (5mL PBS 10x, 45mL DEPC water, 500uL Tween 10%)
Put the dissected material in a microcentrifuge tube containing 500uL of formaldehyde 4% (200uL FA 20% stored at -20°C + 800uL PBT*) for less than 15-20min while dissecting
Once all dissections are done, add 500uL of heptane (small brown bottle in the hazardous storage cabinet) and rotate for 20min
Remove fix, add 500uL methanol
Rotate at maximum speed for 3 min
Remove a much liquid as possible
Wash with methanol 3 times
Keep at –20°C in methanol if necessary.

In situ staining
 Rehydrate in 50%methanol+PBT* for 2min, by adding 500uL of PBT* in a tube containing 500uL methanol
 Remove 500uL, add 500uL of PBT*, wait 2min
 2 washes PBT for 2min
 Fix in 4% FA 15min (200uL 20% FA stored at -20°C + 800umL PBT*) under rotation
 One quick wash PBT
 5 washes PBT 5min each under rotation. During the last wash, separate samples into individual tubes for each staining.
 Incubate in 50% PBT*+HYB for 5min. No rotation when tissues are in HYB solution: be careful, tissues are very fragile when in HYB solution.
 Incubate in 300uL HYB for 5 min
 Incubate in300uL HYB2 (=2mL HYB+80uL salmon testis DNA) for 1h at 55C
 Incubate overnight at 55C with the DIG-labeled RNA probe (1uL in 300uL HYB2, prior to the first utilization, denaturate the probe at 80C for 1min and put immediately on ice)
 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 under rotation
  For larval tissues: incubate for 1h in PBT+goat serum (= 4.8mL milliQ water, 600uL PBS 10x, 60uL Tween 10%, 600uL inactivated goat serum stored at -20°Cin SNC box, top drawer, C6). For embryos: skip this step (otherwise there will be no staining).
 Incubate with anti-DIG-AP (Roche, 1/4000 diluted in PBT for embryos and in PBT+goat serum for larval tissues) for 2h at RT
 3 washes in PBT 10min each
 3 washes in staining solution 10min each

Staining solution: for 10mL
250ul       NaCl 4M
500uL      MgCl2 1M
1mL         Tris HCl 1M
100uL      Tween 10%
8.25mL     milliQ water

Prepare  the NBT/BCIP staining solution: 1mL staining solution + 4.5uL NBT + 3.5 uL BCIP (small brown plastic bottles at -20°C, transparent plastic box, top drawer, C4)
Cover to protect from ligth. From 5min to several hours, wash under the microscope. Staining should appear within 10min.
One quick wash in PBT
3 washes in PBT 10 min


Mounting