The primed in situ labeling (PRINS) methodology, based on rapid annealing of primers followed by in situ elongation for chromosome labeling as proposed by Koch et al. (1) and Gosden et al. (2), could be combined with other methods such as fluorescent in situ hybridization (FISH [3]). The simultaneous visualization of a DNA and a protein target is another goal. The combination of FISH and immunofluorescence offers multiple possibilities for research. However, the simultaneous generation of signals involving DNA and protein staining is difficult. FISH and protein immunostaining already have been described (4), and FISH and indirect immunoflorescence have been combined successfully (5,6). However, despite the widespread use of these techniques, they still require labor-extensive protocol adjustments to achieve correct and satisfactory simultaneous signal detection. Among the problems encountered, there are (1) the persistence of immunological signal after FISH treatments, (2) the permeability of DNA probe after protein fixation, and (3) the antigen durability after DNA denaturation.

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed. Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

As the aim of a study on the chromosome distribution and segregation during the polyploidisation process of megakaryocytes (7) and after some wastage using FISH, we developed a method combining PRINS for labeling of selected centromeres with fluorescent immunostaining of tubulin for visualizing the spindle. We speculated that PRINS would prove more advantageous than FISH for several reasons: (1) the method is rapid, (2) the diffusion of small oligo-nucleotides within the material would be better than it is with classic FISH probes, (3) the prolonged contacts with formamide required during FISH incubation would be avoided, (4) PRINS is highly specific, and (5) PRINS had yet been successively combined with immunochemistry for the study of the organization of repetitive DNA sequences with respect to the kinetochore and for the detection of SV40-like viral DNA and antigens in malignant pleural mesothelioma (8,9).

After quick preliminary attempts, the protocol yielded reproducible results demonstrating the feasibility of PRINS and immunofluorescence. In our hands, it provides an accurate technique to study chromosome anchorage on the spindle and its segregation with respect to metaphase/anaphase "checkpoints" in various physiological or pathological situations in which cells are susceptible to aneuploidization or polyploidization. For a most general point of view, PRINS has proved to be an attractive technology and an excellent alternative to FISH as a means of achieving fluorescent in situ DNA labeling combined with the indirect immunofluorescent tagging of proteins.

Was this article helpful?

0 0

Post a comment