Chromogenic in situ hybridization (CISH) is used to detect specific nucleic acid sequences in tissue, cell, or chromosome aberrations. In the past, in situ hybridization with chromogenic detecion was limited by higher background and low signal intensity with gene specific probes. Invitrogen's patented SPT (Subtraction Probe Technology) utilizes an overlapping assembly of highly specific probe "contigs" whose repetitive ALU and LINE elements have been quantitatively removed by subtractive hybridization, thus preserving unique sequences in the probe contigs. Such technology can be used to determine the presence or absence of a gene, the amplification of a gene, the rearrangement of chromosomes (translocation), or the change in the number of chromosomes. This Tech Tip focuses on common questions associated with CISH.

One step of particular importance is temperature. For specimen preparation, tissue sections should be 4-5 μm and should be baked at 60°C for 2-4 hours before deparaffinization. For heat treatment, it is critical to have the specimen heated at ≥ 98°C but less than 102°C for 15 minutes in the CISH Pretreatment Buffer. This can be accomplished by one of the following: 

  • Pressure cooker: set the timer for 10 minutes. Open the pressure cooker when the pressure has dropped to 0 (Temperature will be around 95°C).
  • Hot plate: heat the CISH Pretreatment Buffer in a beaker; after the buffer temperature is above 98°C, put the slide in and then heat for 15 minutes. 
  • Steamer: make sure the buffer temperature reaches 98-100°C before putting slides in the buffer; then steam for 20 minutes. 


It is important to note that, the time for incubation starts after the buffer is prewarmed, not while the buffer is warming up. For the pepsin digestion equilibrate to room temperature (15-30°C) before applying. A 5 minute incubation time is a good starting point, but optimum digestion time will need to be determined by the user for their respective sample. 

Denaturation, hybridization, and stringent wash follow the same conventions. For denaturation we routinely use 94-95°C for 5 min for the best results. For hybridization, 10-18 hours at 37°C give a signal, which can be done with overnight incubation. For your convenience, Invitrogen carries CISH UnderCover™ Slips which are alternative cover slips that form a heat-resistant, peel-and-stick coverslip for hybridization, are sealed to prevent probe evaporation and tissue specimen dehydration on glass microscope slides, thus eliminating the hassle of messy rubber cement. Once hybridization is complete, CISH UnderCover™ Slips peel off cleanly and quickly. Finally, when using the stringency wash, the temperature should be 70°C. 

One common question that arises is whether or not SPOT-Light probes work with other companies' kits or other companies' probes work with CISH detection kits. The customer is welcome to try any combination of our probes or kits with another company's, but Invitrogen has not verified any of these tests ourselves, and thus cannot make any claims or guarantees. 

Another common question is which detection kit to use. Biotin CISH labeled probes require the CISH Centromere Detection Kit, translocation probes require the CISH Tranlocation Kit which allows for dual staining of probe pairs using bright field microscopy, while DIG CISH labeled probes require the CISH Polymer Detection Kit.