|Choudhary, Ratan - UNIVERSITY OF MARYLAND|
Submitted to: Joint Abstracts of the American Dairy Science and Society of Animal Science
Publication Type: Abstract Only
Publication Acceptance Date: March 12, 2009
Publication Date: July 1, 2009
Citation: Choudhary, R.K., Daniels, K.M., Clover, C.M., Capuco, A.V. 2009. Development of a protocol for staining BrdU-labeled cells within cryosections of bovine mammary tissue that is suitable for subsequent transcriptome analysis. [abstract]. Joint Abstracts of the American Dairy Science and Society of Animal Science. Technical Abstract: Bromodeoxyuridine (BrdU) is a thymidine analog that is incorporated into the DNA of proliferating cells. Immunostaining of BrdU-labeled cells within a histological section requires heat or chemical-mediated antigen retrieval to open the dsDNA and expose the BrdU antigen. We found that in cryosections such treatments, while they do permit staining of the tissue, preclude its use in further gene expression experiments. With cryosections, long antibody incubations and several washing steps, lead to extensive RNA degradation and undesired RNA elution into wash buffers. We have developed a protocol for immunolocalization of BrdU-labeled cells in unfixed cryosections of bovine mammary tissue that does not require harsh DNA denaturation and minimizes RNA degradation and elution into wash buffers. This protocol uses an initial acetone treatment (2 min) followed staining with methyl green (0.5%, 2 min) to stabilize macromolecules, antigen retrieval with deionized formamide (70% in nuclease-free phosphate buffered saline, 5 min incubation), short (10 min) antibody incubations in the presence of RNase inhibitors, and minimal washing, to facilitate recovery of RNA from stained sections. Total RNA was obtained by column extraction (RNeasy Micro kit; Qiagen, Valencia, CA) of cell lysates from sections. Utility of the isolated RNA for gene expression analysis was confirmed using gene-specific primers and quantitative RT-PCR. Furthermore, RNA quality was evaluated by micro-fluidic electrophoresis (Agilent BioAnalyzer) and acceptable RNA integrity numbers (RIN 6.0) were obtained. Staining intensity obtained with this BrdU labeling protocol was similar to that obtained using conventional immunohistochemistry protocols. When coupled with laser microdissection and RNA amplification, this immunostaining protocol should provide a means for evaluating genes expressed by BrdU-labeled cells that are extracted from a complex tissue section.