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Title: A Rapid Method for BrdU Immunostaining in Bovine Mammary Cryosections that Retains RNA Quality

Author
item CHOUDHARY, R - University Of Maryland
item Daniels, Kristy
item Clover, Christina
item Garrett, Wesley
item Capuco, Anthony

Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/30/2010
Publication Date: 6/1/2010
Citation: Choudhary, R.K., Daniels, K.M., Clover, C.M., Garrett, W.M., Capuco, A.V. 2010. A Rapid Method for BrdU Immunostaining in Bovine Mammary Cryosections that Retains RNA Quality. Journal of Dairy Science. 93:2574-2579.

Interpretive Summary: Bromodeoxyuridine (BrdU) is a synthetic nucleotide that mimics the natural nucleotide, thymidine. Consequently, BrdU is incorporated into the cell’s DNA when it is proliferating. Because it can then be detected in tissue sections using antibody-based histological methods, BrdU is a powerful agent for studying various aspects of tissue growth. However, because the staining methods for detecting cells that have incorporated BrdU are harsh, it has not been feasible to obtain good quality RNA from these tissue sections to permit subsequent study of gene expression. We have successfully developed a protocol for detecting BrdU-labeled cells in histological tissue sections that does not destroy the cell’s RNA. Thus, this method provides a means for isolating RNA from BrdU-labeled cells in order to study gene regulation of cell proliferation. Furthermore, the method appears to be applicable for detection of other nuclear protein antigens and so is likely to have broad applicability for studying the regulation of gene expression in specific cells that are isolated from whole tissue.

Technical Abstract: A rapid method of 5-bromo-2’-deoxyuridine (BrdU) immunostaining was developed in cryosections of bovine mammary tissue, while preserving RNA quality of the stained section. BrdU is a thymidine analog that is incorporated into DNA of proliferating cells and thus serves as a proliferation marker. Immunostaining of BrdU-labeled cells within a histological section requires heat, enzymatic or chemical-mediated antigen retrieval to open dsDNA and expose the BrdU antigen. While these established treatments permit staining, they preclude use of the tissue section in further gene expression experiments. Additionally, long antibody incubations and washing steps lead to extensive RNA degradation and elution. We developed a protocol for immunolocalization of BrdU-labeled cells in cryosections of bovine mammary tissue, which does not require harsh DNA denaturation and preserves RNA integrity and quantity. This protocol uses an initial ACEPEG (acetone:polyethylene glycol 300 (9:1 v/v)) fixation (2 min) followed by staining with methyl green (0.5% aqueous, 2 min) to stabilize macromolecules, antigen retrieval with deionized formamide (70% in nuclease-free phosphate buffered saline, 4 min incubation), 5 min antibody incubations in the presence of RNase inhibitors, and minimal washing, to facilitate recovery of RNA from stained sections. We evaluated applicability of this protocol to other nuclear antigens by testing its suitability for two nuclear antigens, estrogen receptor alpha (ERa) and Ki67. In both cases, use of this protocol provided antigen retrieval, good immunostaining and good tissue morphology. However, we did not evaluate RNA quality of ERa- and Ki67-stained sections Quality of the isolated RNA from BrdU-stained sections was evaluated by micro-fluidic electrophoresis and its utility was confirmed using quantitative RT-PCR. Staining intensity obtained with this labeling protocol was similar to that obtained using conventional immunohistochemistry protocols. When coupled with laser microdissection and RNA amplification, this immunostaining protocol provides a means for transcriptome analysis of specific antigen-expressing cells within a complex tissue.