Figure 1 exemplifies typical nuclear phenotypes of DAPI stained progenitor cells (upper panel), monoblast and myeloblast precursor cells (mid panel), and monocytes and granulocytes (bottom panel), represented by xy mid-sections of nuclei acquired with 3D-SIM.
It is worth noting that the ultrathin sections of isolated nuclei present a much cleaner background than that of nuclei in the cell.
To distinguish the total number of nuclei, sections were counter-stained with 1 μg/ml 4', 6-Diamidino-2-phenylindole (DAPI) (Molecular Probes, Eugene, Oregon, USA) in PBS for 1 min. Then the slides were washed 3 times for 5 min each in PBS.
However, tissue sectioning might cause truncation of nuclei, and it has been estimated that when processing single cells from 6 μm sections, truncation leads to a loss of 10 20% of the genetic material [ 33].
(A) 3D-SIM light optical mid-sections from whole 3D acquisitions of nuclei and representative inset magnifications delineating DAPI stained DNA (gray), immuno-stained H3K4me3 (green) and RNA Pol II Ser5P (red).
Thick tissue sections (50 μm) were used for preparation of nuclei, to decrease the likelihood of partial nuclei that would cause an artefactually lowered DNA content.
For example, our analysis reveals that the computation of the image curvature, described in section 3.4.1, may miss a clump of nuclei because of the scale of the fold between two adjacent nuclei.
b f Immunohistochemical staining of tissue sections with nuclei counterstained with hematoxylin.
Together, these results support the possibility that truncation of nuclei during tissue sectioning resulted in loss of genetic information.
We suspected that some of the observed amplification failure and ADO was a result of partial or complete loss of chromosomes as a result of truncation of nuclei during tissue sectioning.
