Embryos for histology and immunohistochemistry were harvested and fixed in 10% neutral-buffered formalin, dehydrated through a graded series of alcohol, embedded in paraffin, sagittally sectioned at 5 μm intervals, and every fifth section was processed for hematoxylin and eosin (H&E) staining according to standard protocols. Remaining sections of wild-type and Ptdsr -/- specimens were used for immunohistochemistry. For detection of apoptotic cells and macrophages, anti-aCasp3 (an antibody specific for activated caspase 3; R&D Systems, Minneapolis, USA) and anti-F4/80 (Serotec GmBH, Düsseldorf, Germany; #MCA 1957) antibodies were used as described by the supplier. Detection was performed using indirect streptavidin with biotinylated secondary antibodies and cobalt-enhanced diaminobenzidine (brown) or fast-red (red) as chromogens. Sections were counterstained with hematoxylin. For whole-mount terminal deoxynucleotidyl transferase-mediated UTP end labeling (TUNEL), limb buds were dissected from E12.5 and E13.5 embryos, fixed in 4% paraformaldehyde and processed for analysis as previously described [50].