4% PFA-Pb 4% Paraformaldehyde in P-Buffer (Prepared Fresh)

EMBRYO FIXATION

DAY 1: Materials

P-buffer … Phosphate-Buffer (1 L)

3.65 g monobasic NaH2PO4 10.93 g dibasic Na2HPO4

4% PFA-Pb … 4% paraformaldehyde in P-buffer (prepared fresh)

- in 125 mL Gibco bottle, heat 70 mL P-buffer to boiling in microwave - weigh 4 g paraformaldehyde powder - place hot buffer in hood (buffer should cool slightly); add paraformaldehyde powder. - add 30 mL P-buffer (cool) - put on ice to cool to 4 oC for use. (Make certain solution cool enough before putting on ice so glass bottle doesn’t crack).

Sucrose solution … embedding solution

30% sucrose (4.5g) in 15 mL 4% PFA-Pb 2

DAY 1: Protocol

Dissection of Embryos

1. Remove embryo completely from placenta sac and place in 6 well plate with 4% PFA-Pb.

2. Put on Orbitter @ RT and fix according to age.

Age of Embryo Volume of Fixative Time for Fixation (mL) (minutes)

E7.5 – E8.5 2 15 E9.5 – E10.5 5 30 E11.5 – E12.5 10 60 E13.5 – E14.5 15 90

3. Replace fixative with sucrose solution and incubate gently on Orbitter @ 4 oC over-night. 3

DAY 2: Materials

- P-buffer

- 50% OCT / P-buffer

- 100% OCT

- place Orbitter @ RT

DAY 2: Protocol

1. CAREFULLY remove embedding solution with vacuum.

2. Add P-buffer to wash; incubate 1 hour @ RT while gently shaking.

3. Replace P-buffer with 50% OCT / P-buffer; incubate 8 hours @ RT while gently shaking.

4. Replace with 100% OCT; incubate over-night @ 4 oC while gently shaking. 4

DAY 3: Materials

- Styrofoam pieces for mounting

- plastic beaker for 2-methylbutane

- stryofoam container for liquid N2

 fill beaker with 2-methylbutane and set in liquid N2 ; cover beaker so it will freeze faster, but don’t freeze solid!

- chill 50 mL centrifuge tubes @ - 80 oC

- Histofreeze

DAY 3: Protocol

1. Label stryofoam pieces.

2. One drop of OCT on stryofoam & freeze. Another drop or two more & freeze.

3. One drop of OCT and orient embryo on top.

4. Squirt OCT underneath and around so embryo will it stick to foam; cover embryo with OCT.

5. Dip embryo into 2-methylbutane (know that it is frozen when bubbling stops). Can leave embryo sitting in this until all samples are done.

6. Put embryo in chilled centrifuge tubes; store @ - 80 oC. 5

Cryostat Sectioning

 Cool everything (e.g., knife) before hand.

Knife …. 4 clamps, 2 hold down & 2 make straight.

Thickness …. 10 M works well; keep 10 M as min. & 20 M as max.

Mounting embryos for sectioning

1. On copper mount, place 1 drop of OCT and position embryo (decide … transverse or sagittal sectioning); freeze for ~ 1 min. (turns very white in colour).

2. Put holder + embryo into stage; screw into place.

3. Use hand roller (outside) to move stage up so embryo just contacts knife.

4. Use brushes to brush things away … DON’T TOUCH!

5. Brush away excess OCT pieces and brush away from knife to keep sharp. 6

IMMUNOHISTOCHEMISTRY

Vector Laboratories (www.vectorlabs.com or phone @ 1-888-629-2121) provide kits.

Mounting frozen sections to slides

1. Sections are air dried. 2. Immediately before staining, fix sections with acetone or appropriate fixative for the antigen under study. 3. Transfer slides into buffer.

ABC Kit

(for sections, also do some samples without 1o antibody as control)

Counterstaining 7