Tm 1000 tabletop sem

Cryosectioned animals were processed after fluorescent in situ development as previously described (Tu et al., 2012 (link)). For nuclear fast red staining, animals were processed for colorimetric ISH and were subsequently fixed overnight in 4% paraformaldehyde (PBS) at 4°C followed by dehydration in 30, 50, and 70% ethanol. Fixed specimens were embedded in paraffin and serial sectioned at 10 μm thickness and counter stained with nuclear fast red.
For scanning electron microscopy, animals were immersed in a relaxant fixative (1% HNO₃, 0.85% formaldehyde, 50 mM MgSO₄) as described elsewhere (Rompolas et al., 2013 (link)) for 5 min and were replaced with fresh fixative and rocked overnight at room temperature. Animals were then transferred to a solution containing 2.5% glutaraldehyde, 2% paraformaldehyde, 1% sucrose, 1 mM CaCl2 in 0.05 M NaCacodylate buffer pH 7.36 and left at 4°C until ready for processing. Animals were rinsed in ultrapure water, and secondary fixation was performed at 4°C overnight in 2% aqueous osmium tetroxide. Samples were dehydrated in a graded series of ethanol and dried in a Tousimis Samdri-795 critical point dryer. Samples were mounted on stubs and sputter coated with gold palladium. Imaging was done with a Hitachi TM-1000 tabletop SEM.

The
morphology of the
ball-milled birch and spruce wood particles was studied with a tabletop
scanning electron microscope (Hitachi TM-1000 Tabletop SEM, Tokyo,
Japan). The acceleration voltage was 15 kV, and the current was between
31.7 and 34.9 μA. 100× and 500× magnifications were
used, and for all samples, the working distance varied between 5760
and 6160 μm. The samples were placed on a carbon tape for imaging
and were not coated.