A Novel Amplification Method of Nonradioactive In Situ Hybridization Signal for Specific RNA with Biotinylated Tyramine.

Pituitary Adenylate-Cyclase-Activating Polypeptide (PACAP) Binding Sites and PACAP/Vasoactive Intestinal Polypeptide Receptor Expression in Human Pituitary Adenomas

“…With this increase in sensitivity, an in situ CARD technique has some advantage over RT-PCR or in situ RT-PCR, including reproducibility and ease of performance of the assay. 18,19 In summary, these studies show a differential distribution of PACAP binding sites and PVR-1, -2, and -3 mRNA expression in pituitary adenomas. The differences observed, especially with regard to prolactinomas, probably reflect different regulatory roles of PVR in these tumors.…”

“…Recent studies have used biotinylated tyramide in a catalyzed reporter deposition ISH (CARD-ISH) amplification system to increase the sensitivity of assays detecting protein by immunohistochemistry 18 and mRNA by ISH. 19 We used reverse transcription polymerase chain reaction (RT-PCR) with Southern hybridization, conventional ISH, and CARD-ISH to analyze the expression of PACAP, PVR-1, -2, and -3 mRNAs, and PACAP binding sites in human pituitary tumors. Our studies localized PVR-1, -2, and -3 in pituitary adenomas and showed for the first time the in situ localization of PACAP receptors.…”

PCR In Situ Amplification and Catalyzed Signal Amplification. Approaches of Higher Sensitive, Non-Radioactive In Situ Hybridization.

“…Thus tyramine is readily conjugated with a variety of hapten labeled esters such as FITC-ester, and biotin-ester by simply mixing the same mole of each component in DMSO [5,6]. Biotin-tyramide and FITC-tyramide have been applied for post ISH amplification after several different ISH methods, including DNA/mRNA ISH and chromosomal ISH with a variety of probe types [9,20,26,30,40,46,47,56,[59][60][61]. In the case of using biotin-tyramide, biotinylated probe is sequentially reacted with primary HRP conjugated streptavidin, 30ug/ml of biotintyramide in the presence of 0.001% H2O2, and secondary HRP conjugated streptavidin (Fig.…”

“…An alternative in situ detection method for rare nucleic acid sequences, termed catalyzed signal amplification (CSA), has been developed [20,26,30,56]. The CSA method is based on the peroxidase catalyzed deposition of phenolic substrates such as p-cresol, tyramine, and homovanillic acid [18].…”

For Consistent Detection of Tissue mRNA in Human Surgical Material by Non-Radioactive In Situ Hybridization Technique.

“…This effect may be ascribed to a rapid fixation of cytoplasmic proteins surrounding cytoplasmic mRNAs, which can contribute to a better preservation of mRNA during tissue processing, tissue storage and the tissue sectioning process. Therefore, this method is, at least, recommendable for the in situ hybridization technique together with high signal amplification method [8].…”