Label IT¢ç Fluorescence In Situ Hybridization Kits

Efficient, direct, non-enzymatic labeling of DNA probes for FISH applications

* Optimized Protocol - Detailed instructions and validation for centromeric and whole chromosome analyses.

* Direct and Indirect Sensitive Detection - Achieve high multicolor sensitivity with optimally labeled DNA probes.

* One-step Chemical Method - Easily and consistently control the labeling reactions.

* Covalent Mechanism - Permanent, non-destructive modification of nucleic acid residues is ideal for FISH; labels do not impact hybridization performance.

Kit Components:
Each FISH Kit includes the appropriate Cy¢ç3, TM-Rhodamine, Fluorescein, or Biotin labeling reagent, solutions and hybridization buffer. Please note that the Label IT¢ç FISH Biotin Kit does not contain Biotin detection reagents.

Figure 1. The Label IT¢ç FISH Kits. The Label IT¢ç FISH labeling reagents are composed of three regions: the label (a fluorophore or biotin) (green), the linker (yellow) which facilitates electrostatic interactions with DNA probes and the reactive alkylating group (blue) that covalently attaches the Label IT¢ç reagents to any reactive heteroatom within the DNA probe. Attachment of the Label IT¢ç reagent to the DNA probe does not alter its structure or affect downstream hybridization performance, and once labeled, the DNA probe can be hybridized to metaphase chromosome spreads and detected.

Figure 2.

Panel A
PAINT analysis with Alu-PCR products from Murine 3T6 somatic cell hybrid (Coriell Cell Repository Mapping Panel 2 - Version 3, Hybrid #NA13140), containing only human chromosome 20. Purified probe DNA was labeled with the Label IT¢ç FISH Cy¢ç3 Kit as recommended in the protocol. 150 ng probe DNA was added to 7.5 µg human Cot-1 DNA and 11 µg salmon sperm DNA before alkaline denaturation. Probe cocktail was ethanol precipitated and resuspended in hybridization buffer and further heat-denatured for 10 minutes at 75¡ÆC prior to hybridization. Metaphase spreads were obtained from PHA-stimulated peripheral lymphocytes. Spreads were RNase A treated and dehydrated prior to denaturing at 72¡ÆC for 3 minutes in 70% Formamide/ 2X SSC, pH 7.5. Dehydrated slides were warmed to 37¡ÆC prior to probe application and sealing in humid chambers. FISH slides were hybridized overnight at 42¡ÆC and washed to remove non-specific signal. Coverslips were removed in 2X SSC and slides were washed three times with 50% Formamide/ 2X SSC, pH 7.5 at 45¡ÆC, for 5 minutes each and then, three times in 2X SSC at 45¡ÆC for 5 minutes each. Hybridized slides were mounted in DAPI/Vectashield for viewing with proper filters. DAPI stained images were overlayed with fluorescent probe dye images.

Panel B
PAINT analysis with Alu-PCR products from Chinese Hamster RJK88 somatic cell hybrid (hybrid #NA10791, see reference above) containing only human chromosome 7. Probe was treated, along with target metaphase spread with the same protocol outlined in "Panel A," except label on probe is generated with the Label IT¢ç FISH TM-Rhodamine Kit.

Panel C
Multicolor PAINT analysis with Alu-PCR products from Chinese Hamster RJK88 somatic cell hybrid (hybrid #NA10791, see reference above), containing only human chromosome 7, and Chinese/Hamster RJK88 somatic cell hybrid (hybrid #NA11580, see reference above) containing only human chromosome 6. Chromosome 7 PAINT was labeled with the Label IT¢ç FISH Biotin Kit and Chromosome 6 PAINT was labeled with the Label IT¢ç FISH Fluorescein Kit. 150 ng of each labeled probe was combined prior to denaturation with the same amount of competitor DNA (Cot-1/salmon sperm) as needed for only 150 ng probe sample. Metaphase spreads were obtained from ATCC Jurkat cells (Clone E6-1) and prepared for probe application as noted in "Panel A." Detection of biotin was obtained with streptavidin-Cy¢ç3 conjugate from Jackson ImmunoResearch Laboratories (West Grove, PA) at 0.01 mg/ml for 1 hour at room temperature after post hybridization washes. 

Figure 3.

Panel D
FISH with alpha-satellite-PCR products from Murine 3T6 somatic cell hybrid (Coriell Cell Repository Mapping Panel 2 - Version 3, Hybrid #NA10791) specific for human centromere 7 and hybrid #NA11580, specific for human centromere 6. Purified centromeric 7 probe DNA was labeled with the Label IT¢ç FISH Cy¢ç3 Kit and centromeric 6 with the Label IT¢ç FISH Fluorescein Kit. 30 ng of each sample probe DNA was added to 1.2 µg human Cot-1 DNA and 18 µg salmon sperm for alkaline denaturation. Probe cocktail and metaphase spread (ATCC Jurkat, Clone E6-1) were treated as noted in "Panel A." Post hybridization, coverslips were removed in 2X SSC and slide was washed three times with 50% formamide/ 2X SSC, pH 7.5 at 45¡ÆC, for 5 minutes each and then, three times in 0.1X SSC at 45¡ÆC for 5 minutes each. Hybridized slides were mounted in DAPI/Vectashield for viewing with proper filters.

Panel E
FISH with alpha-satellite-PCR products from Murine 3T6 somatic cell hybrid (Coriell Cell Repository Mapping Panel 2 - Version 3, Hybrid #NA10791) specific for human centromere 7. Purified centromeric 7 probe DNA was labeled with the Label IT¢ç FISH Biotin Kit. 30 ng sample probe DNA was added to 1.2 µg human Cot-1 DNA and 18 µg salmon sperm DNA before alkaline denaturation. Probe cocktail and metaphase spread (ATCC Jurkat cells, Clone E6-1) were treated as noted in "Panel A." Post hybridization, coverslips were removed in 2X SSC and slide was washed three times with 50% Formamide/ 2X SSC, pH 7.5 at 45¡ÆC, for 5 minutes each and then, three times in 0.1X SSC at 45¡ÆC for 5 minutes each. Detection for biotin was obtained with streptavidin-Cy¢ç3 conjugate from Jackson ImmunoResearch Laboratories (West Grove, PA) at 0.01 mg/ml for 1 hour at room temperature just after post hybridization washes. Hybridized slides were mounted in DAPI/Vectashield for viewing with proper filters.

Panel F
FISH with alpha-satellite-PCR products from Chinese Hamster RJK88 somatic cell hybrid (hybrid #NA10868A, see reference from "Panel A") containing only human chromosome 12. Purified centromeric 12 probe DNA was labeled with the Label IT¢ç FISH TM-Rhodamine Kit. 30 ng of sample probe DNA was added to 1.2 µg human Cot-1 DNA and 18 µg salmon sperm DNA before alkaline denaturation. Probe cocktail and metaphase spread (PHA-stimulated peripheral lymphocytes) were treated as noted in "Panel A." Post hybridization, coverslips were removed in 2X SSC and slide was washed three times with 50% formamide/ 2X SSC, pH 7.5 and 45¡ÆC, for 5 minutes each and then, three times in 0.1X SSC at 65¡ÆC for 5 minutes each. Hybridized slides were mounted in DAPI/Vectashield for viewing with proper filters.