<p> 1</p><p>EMBRYO FIXATION</p><p>DAY 1: Materials</p><p>P-buffer … Phosphate-Buffer (1 L)</p><p>3.65 g monobasic NaH2PO4 10.93 g dibasic Na2HPO4</p><p>4% PFA-Pb … 4% paraformaldehyde in P-buffer (prepared fresh)</p><p>- 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).</p><p>Sucrose solution … embedding solution</p><p>30% sucrose (4.5g) in 15 mL 4% PFA-Pb 2</p><p>DAY 1: Protocol</p><p>Dissection of Embryos</p><p>1. Remove embryo completely from placenta sac and place in 6 well plate with 4% PFA-Pb.</p><p>2. Put on Orbitter @ RT and fix according to age.</p><p>Age of Embryo Volume of Fixative Time for Fixation (mL) (minutes)</p><p>E7.5 – E8.5 2 15 E9.5 – E10.5 5 30 E11.5 – E12.5 10 60 E13.5 – E14.5 15 90</p><p>3. Replace fixative with sucrose solution and incubate gently on Orbitter @ 4 oC over-night. 3</p><p>DAY 2: Materials</p><p>- P-buffer</p><p>- 50% OCT / P-buffer</p><p>- 100% OCT</p><p>- place Orbitter @ RT</p><p>DAY 2: Protocol</p><p>1. CAREFULLY remove embedding solution with vacuum.</p><p>2. Add P-buffer to wash; incubate 1 hour @ RT while gently shaking.</p><p>3. Replace P-buffer with 50% OCT / P-buffer; incubate 8 hours @ RT while gently shaking.</p><p>4. Replace with 100% OCT; incubate over-night @ 4 oC while gently shaking. 4</p><p>DAY 3: Materials</p><p>- Styrofoam pieces for mounting</p><p>- plastic beaker for 2-methylbutane</p><p>- stryofoam container for liquid N2</p><p> fill beaker with 2-methylbutane and set in liquid N2 ; cover beaker so it will freeze faster, but don’t freeze solid! </p><p>- chill 50 mL centrifuge tubes @ - 80 oC</p><p>- OCT</p><p>- Histofreeze </p><p>DAY 3: Protocol</p><p>1. Label stryofoam pieces.</p><p>2. One drop of OCT on stryofoam & freeze. Another drop or two more & freeze. </p><p>3. One drop of OCT and orient embryo on top.</p><p>4. Squirt OCT underneath and around so embryo will it stick to foam; cover embryo with OCT.</p><p>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.</p><p>6. Put embryo in chilled centrifuge tubes; store @ - 80 oC. 5</p><p>Cryostat Sectioning</p><p> Cool everything (e.g., knife) before hand.</p><p>Knife …. 4 clamps, 2 hold down & 2 make straight.</p><p>Thickness …. 10 M works well; keep 10 M as min. & 20 M as max.</p><p>Mounting embryos for sectioning</p><p>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).</p><p>2. Put holder + embryo into stage; screw into place.</p><p>3. Use hand roller (outside) to move stage up so embryo just contacts knife.</p><p>4. Use brushes to brush things away … DON’T TOUCH!</p><p>5. Brush away excess OCT pieces and brush away from knife to keep sharp. 6</p><p>IMMUNOHISTOCHEMISTRY</p><p>Vector Laboratories (www.vectorlabs.com or phone @ 1-888-629-2121) provide kits.</p><p>Mounting frozen sections to slides</p><p>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.</p><p>ABC Kit</p><p>(for sections, also do some samples without 1o antibody as control) </p><p>Counterstaining 7</p>