IN SITU HYBRIDIZATION
In situ hybridization utilizes complementary DNA or RNA to detect and localize a specific DNA or RNA sequence in tissue by high resolution microscopy. FISH is a modification of this technique that uses fluorescent probes, and is often used to detect the chromosome location of specific DNA sequences. Protocols for in situ hybridization usually include chemical denaturation and fixation to hold the targets in place and make them more accessible to hybridization. The hybridization step usually takes place at an elevated temperature (50-80 degrees Celsius), and can be conducted for up to 24 hours before stringent washing to remove non-specific binding.
SecureSeal ™ chambers from Grace Bio-Labs are ideally suited for in situ hybridization assays. This adhesive seal bonds to glass and is temperature resistant even after heating. Reactants may be added and removed through seal-able ports. All materials used in SecureSeal are compatible with fluorescent, radiometric or enzymatic detection methods. Suire et al. recently described using SecureSeal chambers to fix in situ hybridized bone marrow plugs for high resolution imaging analysis. Some of our customers have also use CoverWell Incubation chambers for in situ hybridization (Shintani et al, 2014) and our HybriSlip ™ in place of glass coverslips (del Pilar Gomez, 2011) which can be placed in humid chambers to minimize evaporation. The ProPlate chambers are open at the top, and can be sealed with adhesive sealing strips. A recent paper by Jakt et al. used our CultureWell ™ gaskets for an in situ hybridization protocol that used combinatorial fluorescence detection. The CultureWell gasket was bound to a glass slide to create 8 wells in which cells were cultured and processed in parallel.
- Suire, C, et al. 2012. Isolation of the stromal-vascular fraction of mouse bone marrow markedly enhances the yield of clonogenic stromal progenitors. Blood 119:e86-e95.
- Shintani, M, et al. 2014. Single-Cell analyses revealed transfer ranges of IncP-1, IncP-7 and IncP-9 Plasmids in a Soil Bacterial Community. Applied Environmental Microbiology 80:138-145
- del Pilar Gomez M, et al. 2011. Arrestin in ciliary invertebrate photoreceptors: molecular identification and functional analysis in vivo. J. Neuroscience 31:1811.
- Jakt, ML et al. 2013. A continuum of transcriptional identities visualized by combinatorial fluorescent in situ hybridization. Development 140: 216-225.