In situ hybridization for A-P staining (modified BDGP protocol: J Kearney) Protocol: Embryos for in situ: Embryos should be fixed

in 4% formaldehyde/PBS (see fixation protocol) and washed 3 times in 100% methanol (instead of ethanol) prior to storage at -20C. Embryos can be stored for at least up to one year prior to in situ hybridization. It is recommended that freshly fixed embryos be used when available. Approximately 25ul of embryos is a good amount to use per in situ probe, although more or less can be used. In Site hybridization using DIG-labeled RNA probes: Day 1 (Steps 1-4 no nutation) 1. Remove methanol for formaldehyde-fixed embryos. 2. Rehydrate for 2 minutes in 3:1 Methanol: 4% Formaldehyde/PBS. (750ul/250ul, more specifically 750ul 100% Methanol, 27ul 37% Formaldehyde, 223ul 1x PBS) 3. Rehydrate for 5 minutes in 1:3 Methanol: 4% Formaldehyde/PBS. (250ul/750ul, more specifically 250ul 100% Methanol, 81ul 37% Formaldehyde, 669ul 1x PBS) 4. For ovaries you want to separate the eggs by pipetting up and down slowing using siliconized tips (located in Roketa’s drawer) start from a cut tip progressing to a regular tip until you notice a separation in the ovaries they will eventually break apart. DO NOT discard the tips after use put them back into container for reuse. 5. Fix for 10 minutes in 4% Formaldehyde/PBS. (108ul 37% Formaldehyde, 892ul PBS) 6. Wash 6X in 1X PBT (Tween) (Probably do washes for about a minute each and allow the ovaries or embryos to settle). 7. Add Hybridization buffer without dextran sulfate (500 ul). 8. Rock for 1 hour at room temperature. 9. Dilute probe 1:100 in Hybridization buffer with 5% dextran sulfate (I use 2ul of probe in 200ul of hybridization buffer. 10. Heat probe dilution to 85oC for 5 minutes, place on ice 2 minutes, then place at 55o for 2 minutes. 11. Remove hybridization buffer without dextran sulfate from embryos. Add warmed probe. Incubate O/N at 55oC. Day 2 1. Add 100ul of wash buffer to embryos. Place at 55oC until embryos settle. Remove liquid and wash 2X in 200ul of wash buffer at 55oC. 2. Wash 8X for 30 minutes in 200ul of wash buffer at 55oC. 3. Wash O/N in wash buffer at 55oC. (I have left embryos washing for 3 days at 55oC, in general the longer you wash the more background you remove). Day 3 1. Rinse in 1ml of PBT (Tween). 2. Rock for 30 minutes at RT in 1ml of PBT (Tween); remove PBT.

6. Wash for 5 minutes at RT in 1 ml AP buffer. 5. Place ovaries or embryos on slide. Rinse 2X with 1ml PBT. Rock for 2 hours at RT in 300ul of PBT containing 5% goat serum and 1:2000 dilution of anti-DIG-AP Fab fragments (Roche). The fluorescence reaction takes from 15 minutes to 2hours depending on probe used. 13. When mounting cover slip use a little of fluormount G to place coverslip on slide. To Prep Slides 1. (Can be anywhere from 20 minutes to 5 hours at RT. remove AP buffer. Rock at RT until desired color is achieved. Rinse 3X in PBT to stop color reaction. Add 100uL of anti-dig POD (Roche) at 1:50 in 0. . After step 14 in the above protocol. you can place at 4o C O/N) 10. Rinse 2X with 1ml AP buffer (make up fresh : see below for reagent list and amounts). Place coverslip on slide and using clear nail polish paint cover slip on slide and allow to dry before placing slide in In Situ Slide Box located at 4oC. Add 1ml BCIP/NBT color development solution (3-5ul Bcip and 45ul NBT per ml) 9. Wash 3X 10 minutes in 1X PBT. 12. 4. 8. store at 4oC until ready to mount.5% blocking buffer to each tube of embryos. 2. Rinse 2X in PBT to stop color reaction (1ml about 1 min per wash). 3.5% blocking buffer for 1-2 hours at RT. 4. Add 200ul DAPI solution (cover tube with aluminum foil) and rock for 10 mins at room temp. If the color has not developed by 2 hours. 5 ml AP Buffer: 500uL/100 mM Tris 250uL/50 mM MgCl2 100uL/100 mM NaCl 0. Wash 9X for 10 minutes with 1ml PBT (at RT and rock).15 mL SDH2O Modifications: This protocol can be adapted for fluorescence with the Perkin Elmer TSA kit. 11. add 500uL of 0.3. Let sit at RT for 30 minutes.5 uL/ Tween (0. 7.01%) 4. Add 70% glycerol. Vacuum up remaining liquid. Add 50 uL of Cy3 tyramide (Perkin Elmer TSA kit) at 1:50 in amplification diluent (also supplied by kit).

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