Troubleshooting immunohistochemical labelling of proliferating cell nuclear antigen (PCNA) in cryocut tissue sections of mouse prostate

Journal of Pharmacological and Toxicological Methods
Jin Han XieSabatino Ventura

Abstract

The use of antibodies to proliferating cell nuclear antigen (PCNA) for the immunohistochemical detection of proliferating cells has been limited in frozen tissue sections because of its temperature dependence and instability in formaldehyde. In this study various protocols for the immunohistochemical staining of PCNA in frozen mouse prostate tissue sections were tested in order to identify optimal conditions. Fresh prostate tissues from 8 week old mice were frozen in liquid nitrogen or fixed with formaldehyde or paraformaldehyde before freezing with liquid nitrogen. Frozen tissues were then cut in a cryostat and unfixed sections were fixed by dipping slides with sections into fixative. Slide-mounted tissue sections were permeabilized with Triton X-100 before incubating overnight in primary antibody to PCNA diluted to different concentrations in different diluting media at room temperature or 4 degrees C. Secondary antibody was applied in the same medium as the primary. 19 different experimental protocols were examined. Only one protocol showed strong positive immunostaining for PCNA. PCNA-immunopositive cells were observed in greater abundance in the stromal layer. Paraformaldehyde fixation with Triton X-100 permeabilization wi...Continue Reading

Citations

Feb 27, 2010·Journal of Pharmacological and Toxicological Methods·Michael J Curtis
Oct 17, 2017·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·Zienab A GoudaSamia Hussein

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