MCAs from male Sprague-Dawley rats were carefully dissected from the brain as described above, fixed in 4% paraformaldehyde in phosphate buffer for 1 h, followed by rinsing in Soerensen's phosphate buffer with increasing concentrations of sucrose.
The scores for each individual parameter were then added together to give a total score between 0 and 12. Colonic specimens obtained as described were fixed in formalin for at least 24 hours, embedded into paraffin, and cut into 4 5 µm sections.
All additional evaluations were described and fixed in the statistical analysis plan before database closure.
Whole-mounts were prepared as described and fixed in acetone for 2 hours, then rinsed in PBS (3×30 minutes).
After 3 days in culture, cells were surface-stained as described above, fixed in 1% paraformaldehyde on ice for 20 min, and stored at 4°C overnight.
At 7 days after injury, tissue samples were collected as described and fixed in freshly made 4% PFA with 0.01% glutaraldehyde (Sigma) in PBS for 3 hours.
For evaluation of lung metastases, lungs were excised from tumor bearing experimental animals as described above, fixed in 10% neutral-buffered formalin, then paraffin embedded.
The brains were dissected as described above, fixed in 4 % formaldehyde overnight and processed according to standard protocols for the preparation of paraffin-embedded tissue blocks.
For examination of tumour pathology, "gut roll" preparations were made from mice with overt signs of neoplasia as described above, fixed in 10% buffered formalin, wax embedded, and stepped serial sections stained with haematoxylin and eosin.
Whole-mount mammary glands, prepared as described [27], were fixed in Tellys fixative (100 ml EtOH 70%, 5 ml formalin, 5 ml glacial acetic acid), rehydrated and stained with hematoxylin for 3 h.
