In situ hybridization (ISH) is a technique that uses nucleotide probes targeting the sequence of interest to hybridize to locate and visualize specific sequences. This method allows RNA or DNA molecules to be seen in a fixed cell or tissue. ISH can be widely used to study infectious agents, cancer or developmental biology.
ISH is a powerful technology. Creative Biolabs provides you with general procedures and tips for performing ISH, specifically including protocols, material support and a set of useful troubleshooting tips. Any laboratory scientist can easily use it for fast and efficient detection of gene of interest expression and mRNA location.
|Stages||Solutions and Reagents|
|Sample Preparation||Washing buffer, fixative, coating solution|
|Pretreatment||Protease, hydrochloric solution, blocking buffer, washing buffer|
|Hybridization||Hybridization solution, probe solution, washing solutions|
Common sample types for ISH include cells and tissues. Cell samples may include smears, cell debris, cell pellets, etc. Tissue sections may include frozen, paraffin sections, etc. Select the appropriate fixative for the sample. Ideally, ISH fixation should preserve both RNA and DNA as well as tissue morphology. Pre-treat the slide with a suitable coating solution to ensure that the tissue section adheres to the glass slide.
A series of preprocessing steps prior to hybridization can improve the efficiency of hybridization and reduce non-specific background staining. It is necessary to first permeabilize the sample. Treat with protease to increase the accessibility of the target by digesting the proteins surrounding the target nucleic acid. Combining this with other unmasking techniques such as treatment with sodium bisulfite, sodium thiocyanate or hydrochloric acid increases the hybridization signal. Certain tissues have endogenous biotin or alkaline phosphatase (AP). Use blocking agents to inhibit non-specific binding.
Specificity, sensitivity, ease of tissue penetration, stability of the hybrid and reproducibility of the technique must be considered when selecting the best probe for ISH. Select labeling molecules with relatively high specific activity and morphological resolution, such as biotin, fluorescein, and other non-isotopic labels.
Dilute the probe with the hybridization solution, heat to denature the probe and cool it immediately on ice to prevent re-annealing. At the desired hybridization temperature, add the diluted probe to the slide covering the entire sample and incubate in a humidified hybridization chamber. Optimize the hybridization temperature based on the sequence of the probe used and the sample type.
After hybridization, remove unbound probes or probes that bind incompletely matched sequences with washing solutions. Perform washes under stringent conditions close to where hybridization occurs. Control solution parameters such as temperature, salt and detergent concentration to eliminate non-specific interactions.
We can demonstrate the presence of a probe-target hybrid by detecting the probe label molecule. If you use isotopically labeled probes, they can be detected by photographic film or photographic emulsion. If you use directly fluorochrome or enzyme-labeled probes, they can be detected directly by instant fluorescence microscopy or by incubation in a substrate solution, respectively.
Are you having trouble with your experiments? We are committed to achieving success in your experiments. Check out our troubleshooting tips for common problems in ISH experiments.
No or weak signals
Tissue loss or tissue morphology degraded
Signal strength variation
We can complete the entire ISH protocol quickly, producing highly consistent results. We can likewise develop and validate assays to meet your specific needs. For all questions and concerns regarding any of our products and services, our technical support team is ready to assist you.
For research use only. Not intended for any clinical use.