title: Technical note: A rapid method for 5-bromo-2'-deoxyuridine (BrdU) immunostaining in bovine mammary cryosections that retains RNA quality.
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. A thymidine analog that is incorporated into DNA of proliferating cells, BrdU serves as a proliferation marker. Immunostaining of BrdU-labeled cells within a histological section requires heat, enzymatic or chemical-mediated antigen retrieval to open double-stranded DNA, and exposure to the BrdU antigen. Although these established treatments permit staining, they preclude use of cells within the tissue section for further gene expression experiments. Additionally, long antibody incubations and washing steps lead to extensive RNA degradation and elution. A protocol was developed for immunolocalization of BrdU-labeled cells in cryosections of bovine mammary tissue, which does not require harsh DNA denaturation and preserved RNA integrity and quantity. This protocol used an initial acetone:polyethylene glycol 300 [9:1 (vol/vol)] 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), antibody incubation in the presence of RNase inhibitors (5 min), and minimal washing to facilitate recovery of RNA from cells from the stained sections. Applicability of this protocol to other nuclear antigens was evaluated by testing its suitability for staining estrogen receptor alpha and Ki-67 antigen. In both cases, use of the protocol provided good immunostaining and tissue morphology. The RNA quality of estrogen receptor alpha- and Ki-67-stained sections was not evaluated. Quality of the isolated RNA from BrdU-stained sections was evaluated by micro-fluidic electrophoresis and its utility was confirmed using quantitative reverse transcription-PCR. Staining intensity obtained with this labeling protocol was similar to that obtained using conventional immunohistochemistry protocols. When coupled with laser microdissection and RNA or cDNA amplification, this immunostaining protocol provided a means for future transcriptome analysis of BrdU-labeled cells within a complex tissue.
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, College Park 20742, USA.
authors.forename: R K
authors.initials: RK
authors.lastname: Choudhary
authors.forename: K M
authors.initials: KM
authors.lastname: Daniels
authors.forename: C M
authors.initials: CM
authors.lastname: Evock-Clover
authors.forename: W
authors.initials: W
authors.lastname: Garrett
authors.forename: A V
authors.initials: AV
authors.lastname: Capuco
mesh_terms: D000818:Animals; D015415:Biomarkers; D001973:Bromodeoxyuridine; D002417:Cattle; D049109:Cell Proliferation; D016616:Cryoultramicrotomy / Q000379:methods*; D004247:DNA / Q000378:metabolism; D005260:Female; D005404:Fixatives; D015870:Gene Expression; D007150:Immunohistochemistry / Q000379:methods; D008321:Mammary Glands, Animal / Q000648:ultrastructure*; D012313:RNA / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D005404:Fixatives; D012313:RNA; D004247:DNA; D001973:Bromodeoxyuridine