Sensitive Multicolor Fluorescence In Situ Hybridization Using Catalyzed Reporter Deposition (CARD) Amplification

Abstract

SUMMARYWe describe the simultaneous localization of DNA sequences in cell and chromosome preparations by means of differently fluorochrome-labeled (AMCA, FITC, TRITC) tyramides using the catalyzed reporter deposition (CARD) procedure. For this purpose, repeated as well as single-copy DNA probes were labeled with biotin, digoxigenin, and FITC, hybridized, and visualized with three different cytochemical detection systems based on horseradish peroxidase conjugates. These were sequentially applied to interphase nuclei and metaphase chromosomes at low concentrations to prevent crossreaction and nonspecific background. In situ localized peroxidase activity was visualized by the deposition of fluorochrome-labeled tyramide molecules. To allow specific deposition of a second and a third tyramide conjugate for multiple-target fluorescence in situ hybridization (FISH), remaining peroxidase activity was always completely inactivated by a mild acid treatment before application of the next peroxidase conjugate. The CARD reactions were optimized for maximal signal-to-noise ratio and discrete localization by tuning reaction time, H 2 O 2 , and tyramide concentrations. For both repeated and single-copy DNA targets, high FISH signal intensities were obtained, providing improvement of sensitivity over conventional indirect detection systems. In addition, the fluorescence CARD detection system proved to be highly efficient and easy to implement in multiple-labeling studies, such as reported here for FISH.

P16INK4A immunostaining is a strong indicator for high‐risk‐HPV‐associated oropharyngeal carcinomas and dysplasias, but is unreliable to predict low‐risk‐HPV‐infection in head and neck papillomas and laryngeal dysplasias

“…The probes were detected by subsequent application of mouse anti-digoxigenin (Sigma, St. Louis, MO), peroxidase-conjugated rabbit antimouse and peroxidase-conjugated swine anti-rabbit (both DAKO), and visualized by a peroxidase based amplification reaction using rhodamine-labeled tyramide. 35,36 Preparations were mounted in Vectashield (Vector Laboratories, Burlingame, CA) containing 4,6-diamidino-2-phenyl indole (DAPI; Sigma: 0.2 g/ml). Microscope images were recorded with the Metasystems Image Pro System (black and white CCD camera; Sandhausen, Germany) mounted on top of a Leica DM-RE fluorescence microscope equipped with DAPI and rhodamine filters.…”

Identification of Chromosome 9 Alterations and p53 Accumulation in Isolated Carcinoma in Situ of the Urinary Bladder versus Carcinoma in Situ Associated with Carcinoma

“…20,21 In short, the biotin-labeled probe was detected with peroxidase-conjugated avidin (Av-PO, 1:50 dilution; DAKO). Then the first reaction amplification step was performed by applying 50 l of rhodamine-labeled tyramide (1:500 diluted from a 1 mg/ml stock solution in ethanol) in PBS containing 0.1 mol/L imidazole, pH 7.6, and 0.001% H 2 O 2 under a coverslip for 10 minutes at 37°C.…”

Quantitative real-time PCR does not show selective targeting of p14ARF but concomitant inactivation of both p16INK4A and p14ARF in 105 human primary gliomas

“…A rhodamine centromere probe for chromosome 9 (pMR9a) (Rocchi et al, 1991) was separately denatured and mixed with the cosmid probe prior to hybridization. Touch preparations from frozen tumor material were made on glass slides and used for FISH analysis as previously described (Gortz et al, 1999;Speel et al, 1997). Hybridization signals were scored using a Zeiss Axiophot Fluorescence microscope and tri-color images were captured using the Vysis Quips Genetic Workstation (IG Instrumenten-Gesellschaft, Bern, Switzerland).…”