TY - JOUR
T1 - Comparison of immunologic amplification vs enzymatic deposition of fluorochrome-conjugated tyramide as detection systems for FISH
AU - Macechko, P. Timothy
AU - Krueger, Lisa
AU - Hirsch, Betsy A
AU - Erlandsen, Stanley L.
PY - 1997/3
Y1 - 1997/3
N2 - Using various sizes and dilutions of hapten-conjugated DNA probes, we compared catalyzed reporter deposition (CARD) to fluorochrome-conjugated antibody layering (immunological method) for amplifying FISH signals. Cosmid and phage probes that contained human DNA inserts of 40 KB and 15 KB, respectively, and were mapped to chromosome 15q11.2 were used to evaluate these amplification methods. The probes were used either at standard concentrations (10 ng/μl) or at dilutions up to 1:40 (0.25 ng/μl). Detection of FISH signals using either immunological (three antibody layers) or CARD methods were comparable when the undiluted (10 ng/μl) or 1:4 dilution (2.5 ng/μl) of the cosmid probe was used. Use of a single fluorochrome-conjugated antibody layer produced very weak FISH signals. However, addition of an unlabeled secondary antibody followed by a third antibody conjugated to the same fluorochrome (i.e., two rounds of amplification) produced a strong signal that was detected at a 1:4 probe dilution but was not successfully detected at probe dilutions of 1: 10 or greater. In contrast, intense probe signals were produced with the CARD method at all probe dilutions, particularly when coupled to extended hapten- antibody incubation times. The 15-KB phage probe was difficult to detect at a 1:4 dilution with the standard immunologic amplification methods but was readily detected with the CARD method. These data suggest that CARD may be useful for FISH in that (a) less probe may be needed and therefore valuable probe reagents may be conserved, and (b) smaller targets may be detected, thus extending the range of this technique.
AB - Using various sizes and dilutions of hapten-conjugated DNA probes, we compared catalyzed reporter deposition (CARD) to fluorochrome-conjugated antibody layering (immunological method) for amplifying FISH signals. Cosmid and phage probes that contained human DNA inserts of 40 KB and 15 KB, respectively, and were mapped to chromosome 15q11.2 were used to evaluate these amplification methods. The probes were used either at standard concentrations (10 ng/μl) or at dilutions up to 1:40 (0.25 ng/μl). Detection of FISH signals using either immunological (three antibody layers) or CARD methods were comparable when the undiluted (10 ng/μl) or 1:4 dilution (2.5 ng/μl) of the cosmid probe was used. Use of a single fluorochrome-conjugated antibody layer produced very weak FISH signals. However, addition of an unlabeled secondary antibody followed by a third antibody conjugated to the same fluorochrome (i.e., two rounds of amplification) produced a strong signal that was detected at a 1:4 probe dilution but was not successfully detected at probe dilutions of 1: 10 or greater. In contrast, intense probe signals were produced with the CARD method at all probe dilutions, particularly when coupled to extended hapten- antibody incubation times. The 15-KB phage probe was difficult to detect at a 1:4 dilution with the standard immunologic amplification methods but was readily detected with the CARD method. These data suggest that CARD may be useful for FISH in that (a) less probe may be needed and therefore valuable probe reagents may be conserved, and (b) smaller targets may be detected, thus extending the range of this technique.
KW - CARD
KW - FISH
KW - human chromosomes
KW - immunological amplification
KW - tyramide amplification
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U2 - 10.1177/002215549704500303
DO - 10.1177/002215549704500303
M3 - Article
C2 - 9071317
AN - SCOPUS:0031000109
SN - 0022-1554
VL - 45
SP - 359
EP - 363
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
IS - 3
ER -