Solutions:

4%Paraformaldehyde (PFA)/PBS (100ml)

0.2g NaOH
4g PFA (wear mask to weigh)
0.84g NaH₂PO₄•H₂0

• heat while stirring until dissolved
• chill on ice
• gravity filter through fluted whatman paper and return to ice until use
• make fresh the day of use
• check pH (with pH paper) to 7.2

18% Sucrose (100ml)

18g sucrose
10ml 10X DPBS (Biowhittaker)
H₂0 to 100ml

• filter sterilize and store at 4°C
• make fresh the day of use

Procedure:

A. Embryos

1. Dissect embryos from a GFP homozygous mating free of the uterine muscle and dicidua at 8.5 and 9.5 dpc (0.5 dpc on day of plug). Fix embryos in 4% PFA for 1hr at 4¡ãC.
2. Wash several times in PBS and mount in PBS.
3. Analyze on day of isolation and fixation.

1. Anesthetize and perfuse adult mice with 20ml cold PBS followed by 25ml cold 4%PFA.
2. Isolate tissues (heart, brain, liver, spleen, kidney, and lung) and submerge in 4% PFA overnight at 4¡ã with slight agitation.
3. Following isolation, rinse tissues with several changes of PBS for 1 hour each at 4¡ãC and then transfer to 18% sucrose at 4¡ãC.
4. After tissues have equilibrated (24-36 hours), embed in freezing medium (TBS, Durham, NC), and freeze in 2-methyl-butane submerged in liquid nitrogen.
5. Store at -80¡ãC until use.
6. Embedded tissues were equilibrated to -20¡ãC and 60-100¦Ìm sections were cut using a Leica CM3000 cryostat.

Notes: In our lab GFP was visualized in both embryos and adult tissues using a Leica TCS SP laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany). Optical sectioning was performed along the z-axis. Projections of the z-stacks were generated using NIH image 1.62b7+3Dview 1.01 software (National Institutes of Health, Bethesda, MD) and composite images were assembled in Adobe Photoshop 5.5 (Adobe Systems, Inc., San Jose, CA). }