Figure 4.

The nuclear matrix retains the RNP network of the unextracted nucleus with good structural preservation. CaSki cells were prepared for conventional thin section microscopy before (A) and after (B) the removal of chromatin by the cross-linking/DNase I procedure. Thin sections were selectively stained for RNA by the EDTA regressive staining procedure (14) to visualize the RNP network that is an important part of the nuclear matrix. The nuclear lamina (L) forms the periphery of both nucleus (A) and nuclear matrix (B). The removal of chromatin after formaldehyde cross-linking did not substantially alter the structure or spatial distribution of the nuclear RNP network. (C) At higher magnification interchromatin granule clusters, sites of RNA-splicing factor concentration, could be seen with good preservation in the RNP network of the cross-link stabilized nuclear matrix. The CaSki nuclear matrix in this panel was counterstained with the B4A11 antibody which recognizes an RNA splicing factor and with a colloidal gold-conjugated second antibody. This comparison of RNP network ultrastructure in the nucleus and cross-link stabilized nuclear matrix showed good preservation of architecture. The RNP network was not substantially altered by the removal of chromatin if the structure was first extensively cross-linked. [Bars = 500 nm (A and B) and 200 nm (C).]