For the pregnant groups, four placentas and 4 fetuses were also collected from each pregnant mouse, of which half were frozen immediately and the other half were fixed in formalin for further study.
Some lungs from both groups were frozen and stored in liquid nitrogen immediately after collection until RNA extraction, and the remaining were fixed in formalin for further histopathology evaluation.
Placentas were separated in two halves, one half was frozen for RNA extraction and the other was fixed in 10% formalin for further processing.
Cells were fixed in 1% PFA before further analyses.
After sacrifice, femur sections were fixed in 4% paraformaldehyde and further decalcified for 4 weeks in a solution made up of 10% formic acid plus 5% paraformaldehyde.
Immediately after resection, one half of each tumor was cryogenized into liquid nitrogen whereas the other part was fixed in 4 % formalin and further used for immunohistological analyses.
For immune electron microscopy, kidney samples were fixed in 8% formaldehyde in PBS, further processed in sucrose and glycine before mounted and immersed in liquid nitrogen as described [48].
Pieces of tumor pancreata were fixed in 4% (wt/vol) formaldehyde for further immunostaining analysis or prepared for RNA extraction.
For the analysis of axonal length, neurons were transfected with GFP-plasmids after 2 days in vitro and were fixed in 4% paraformaldehyde after a further 24-hour incubation.
Adult worms were identified by direct light microscopy while still alive; those that needed further examination were fixed in 10% formalin and stained with Semichon acetocarmine, mounted on a slide, and measured (18 ).
