In situ hybridization with polymerase chain reaction-derived single-stranded DNA probe and S1 nuclease

Abstract

A rapid and simplified protocol for in situ hybridization (ISH) with polymerase chain reaction (PCR)-derived single-stranded DNA probes and S1 nuclease revealed transcripts of bone matrix proteins on decalcified skeletal bone specimens. Mouse bone tissue was fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin. Each pair of primers for reverse transcriptase -PCR was designed to amplify a 280-bp DNA fragment from the coding region of the mature protein of mouse osteonectin (ON) and a 320-bp fragment from the coding region of mouse osteopontin (OP). Initial PCR products were eluted, purified, and reamplified by unidirectional PCR in the presence of the digoxigenin (DIG)-labeled dUTP. ISH was carried out by proteinase K treatment, hybridization, and washing. The unhybridized single-stranded DNA probe was selectively removed by S1 nuclease treatment. Hybridized probes were visualized with the alkaline phosphatase-conjugated anti-DIG antibody. The transcripts of ON and OP were clearly detected on the thin sections of the decalcified bone. Because this protocol does not require cloning or in vitro transcription, reliable and stable ISH can be done in an ordinary laboratory equipped with a thermal cycler.

Gene Expression during Fracture Healing in Normal Mouse: In Situ Hybridization on Hard Tissues for Investigation of Regenerative Mechanisms.

“…The hyaline cartilage of the growth plate was gradually replaced by bone tissue and decreased in its thickness, resulting in longitudinal growth of the long bone. ted onto the nylon filter, and no cross reactivity was found as previously described [21]. For negative controls, DIG-labeled sense probes were generated with sense primer-primed uni-directional PCR.…”

“…A sequential change of their expression responding to endogenous and exogenous signals can be detected earlier by in situ hybridization (ISH) than by immunohistochemistry. We synthesized single-stranded DNA probes, more stable than RNA probes, by uni-directional PCR without in vitro transcription [21]. Furthermore, RNA of mouse bone tissue of the fracture model fixed with 4% PFA for 2 days was as well preserved after decalcification with 20% EDTA for 4 days as Morse's solution [32].…”

Expression of Bone Morphogenetic Proteins (BMPs) in Fractured Mouse Bone Tissue: In Situ Hybridization with Polymerase Chain Reaction (PCR)-derived Antisense DNA Probe.

“…We employed single-stranded antisense DNA probes synthesized by uni-directional PCR [9]. Among various nucleic acid probes recently introduced, DIG-labeled single-stranded RNA probes transcribed in vitro from linealized double-stranded DNA template are commonly used [18].…”

Parathyroid Hormone-related Peptide-producing Multiple Myeloma and Renal Impairment

“…Final development of the sections was carried out with 3,3'-diaminobenzidine containing 0.03% H2O2. ISH was performed using formalin-fixed, paraffin-embedded tissue sections with a digoxigenin (DIG)-labeled single-stranded DNA probe for PTHrP, as previously described (3,9,10).…”