Reagent Preparation:

Prepare all reagents fresh using RNase-free conditions.

Cresyl Violet Acetate Staining-

Use disposable mTubs (Erie Scientific cat# TR-MTUB) during staining for all reagents except stain and xylene steps.

1. 75% Ethanol in nuclease-free water- 400 ml (Use sterile 750 ml plastic flask)
   300 ml Absolute Ethanol
   100 ml Nuclease-free water
2. 95% Ethanol in Nuclease-free water- 200 ml (Use sterile 250 ml plastic flask)
   190 ml Absolute Ethanol from fresh bottle.
   10 ml Nuclease-free water
3. 100% Ethanol from fresh bottle.
4. 1% Cresyl Violet acetate (CVa) 50 ml
   0.5 gram Cresyl Violet acetate (Sigma-Aldrich, cat# C1791)
   50 ml Nuclease-free water in sterile conical tube.
   Vortex tube with stain for 15 seconds and set aside to settle.
5. Sterilize a glass stain dish with Rnase Zap and rinse with nuclease free water. Blot with Kim wipes and place under the hood to completely dry. Use this dish for the xylene steps during deparaffinization.

Just before staining, pull 10 cc of 1% CVa into a sterile syringe.
Fit syringe tip with a 0.22 µm filter (Millipore, cat# SLGS033SS)

Lysis Buffer Reconstitution-

Arcturus DNA Pico pure® Kit (Molecular Devices cat# KIT0103). Record date on kit as received. Prepare individual lysis buffer fresh every time.

1. Pipette 155 µl of Reconstitution Buffer (2 ml vial) into 1 Proteinase K buffer (0.5 ml) vial and date.
2. Gently vortex to mix the reagents and place tube on ice. Avoid prolonged mixing as this may denature proteinase K.
3. Discard any buffer not used in one day.

Slide Preparation:

Sectioning of PFPE embryo blocks:

1. Prepare auto-microtome (Leica RM2265) for Rnase-free conditions.
2. Cut serial sections at 6 µm until you see a good whole embryo section and call Julie to verify. Collect all sections, two sections per charged slide.
3. Once desired area is reached (middle of embryo – representative section), collect 1 section on a charged slide for H&E.
4. Change section thickness to 8 µm.
5. Cut 9 serial sections placing 3 sections per sterile PEN foil slide for LM.
6. Cut and perform LM within one week. Store slides at room temperature.
7. Change section thickness back to 6 µm.
8. Cut another section for H&E on a charged glass slide.
9. Change section thickness to 8 µm.
10. Cut 9 serial sections placing 3 sections per sterile PEN foil slide for LM. These two sets will be used for LM and each set will go into a separate 500 µl lysing tube.
11. Change section thickness back to 6 µm.
12. Cut section for H&E on a charged glass slide.
13. Repeat steps 9 through 12 to obtain a third set for back up. If these slides are not used within 1 week of sectioning, discard.
14. After all sections from all study blocks have been obtained for LM, return to blocks and continue to section through the embryo at 6 um per section. Collect 2 sections per charged slide. Please use care in re-facing block.

LM Staining:

1. Deparaffinize in 2 fresh xylene baths (use xylene that has not been opened more than 3 months) for 4 minutes each.
2. Clear in 100% ethanol for 30 seconds.
3. Rinse in 95% ethanol for 30 seconds.
4. Rinse in 75% ethanol for 30 seconds.
5. Rinse in nuclease-free water for 30 seconds.
6. Apply drops of cresyl violet acetate to cover each tissue section on slide and stain for 30 seconds.
7. Rinse in 75% ethanol for 30 seconds.
8. Rinse in 95% ethanol for 30 seconds.
9. Rinse in 100% ethanol for 30 seconds. Repeat.
10. Air dry slides, with tissue up, on a clean Kim wipe under hood for 5 minutes.
11. Prepare Leica LMD for microdissection.

Laser Microdissection:

Perform LM using the Leica AS/LMD UV laser microdissection instrument.

1. Label a sterile 500 µl microfuge tube and attach to LMD holder just before microdissection of sample.
2. 50 µl of reconstituted Arcturus Pico pure DNA lysing buffer is placed in the microfuge tube top. Pipette carefully to avoid bubbles.
3. Embryo is identified and microdissected from the 9 serial sections using predetermined laser parameters.
4. Remove microfuge tube from LMD holder and continue with DNA isolation.
5. Keep all tubes inverted at room temperature until LM is complete.

DNA Isolation:

1. Tubes with microdissected material and lysing buffer are left upside down and placed in the bottom of the Arcturus heat block (amber colored) tray.
2. The pre-heated heat block top is carefully placed on top of tubes in tray and the whole unit is placed in a preheated 65°Coven overnight (18-24 hr).
3. Remove tubes and spin at 1000x g.
4. Place in 95°C preheated/shaker unit for 10 minutes to inactivate lysing buffer.
5. Quickly place samples in a cardboard freezer box, labeled with investigator’s name for storage at -20°C until pick-up.