In Situ Hybridization Services
In Situ Hybridization Histochemistry (ISHH) or In Situ Hybridization (ISH) for short, refers to localize a specific DNA or RNA sequence in tissue sections / cells using a labeled complementary DNA or RNA strand (i.e., probe). There are three kind of ISH depends on the probes and the target, i.e., DNA-DNA hybridization, DNA-RNA hybrization and RNA-RNA hybridization. Other than the IHC to detect the target proteins, ISH can be used to mark the chromosome structures. Fluoresence In Situ Hybridization (FISH) is often used for finding specific features of DNA in tumorgenesis, developmental biology, genetic counselling, medicine, and species identification.
ALV provides the ISH/FISH services.
Service Description
1. Diagnosis of tumors and detect viruses,
2. Detect the gene expression in tissues and cells,
3. Co-operating research project,
4. Optimize conditions of ISH for customers.
Procedure
Preparation of Material.
The most common tissue sections used with in situ hybridization are
a) Frozen sections. Fresh tissue is snap frozen (rapidly put into a -80 freezer) and then when frozen embedded in a special support medium for thin cryosectioning. The sections are lightly and rapidly fixed in 4% paraformaldehyde just prior to processing for hybridization.
b) Paraffin embedded sections. Sections are fixed in formalin as one would normally fix tissues for histology and then embedded in wax (paraffin sections) before being sectioned or
c) Cells in suspension. Cells can be cytospun onto glass slides and fixed with methanol
Choice of Probes
Probes are complimentary sequences of nucleotide bases to the specific mRNA sequence of interest. These probes can be as small as 20-40 base pairs or be up to 1000 bp. Although ultimately the question you ask and the type of sequence you are trying to detect is the overriding factor, one needs to optimize, as much as possible the conditions one uses. The strength of the bonds between the probe and the target plays an important role. The strength decreases in the order RNA-RNA to DNA-RNA. This stability is in turn influenced by the various hybridization conditions such as concentration of formamide, salt concentration, hybridization temperature, and pH.
In Situ Hybridization HistoChemsitry (Using DIG-cDNA as probe for example)
Material Treatments
● 0.1M PBS (pH 7.2) for 5-10min,
● 0.1M Glycine/0.1M PBS for 5min,
● 0.3%TritonX-100/0.1M PBS for 10-15min,
● 0.1M PBS Wash 5min×3 times, add Protein Kinase K(1μg/ml)，37℃ incubate for 30 min,
● 4%PFA for 5min,
● 0.1M PBS Wash 5min×2 time, immerse in fresh prepared 0.25% acetic anhydride/0.1M Triethanolamine for 10min,
● PreHybridization: Dropwise add pre-hybridization solution, 42℃ for 30 min。
● Hybridization: Pour out the pre-hybridization solution, dropwise add 10-20μl hybridization solution (dilute the denatured probed in pre-hybridization solution, 0.5 ng/μl ), covered by cover slips or paraffin wax, 42℃ overnight, ( DO NOT Forget the Negative Controls)
● Rinse: (1) 4×SSC、2×SSC、1×SSC、0.5×SSC 37℃ 20min each; 0.2×SSC 37℃ for 10min; 0.2×SSC and 0.1M PBS mixture (1:1) for 10min; 0.05M PBS 5min×2 times,
● 3% BSA/0.05M PBS coating, 37℃ for 30min,
● Dropwise add antidigoxin-antiSAP (serum alkaline phosphatase) complex (starting dilution 1:5000), 4℃ overnight,
● 0.05M PBS 15min×4 times; TSM1 10min×2 times; fresh prepared TSM2 10min×2 times,
● Developing: Dropwise add delevoper to the slides, 4℃ in dark box overnight,
● Dip the slides in TE for 10-30min to stop the reaction,
● Gradient dehydration using alchol; derosination dymethylbenzene,
● Sealing the slides for microscopic inspection.
1. Climbing Cell Slides for ISH
Solution I: ·Customer provides the treated cells and probes (includes the User Manual). Crnbio follows the protocols described above.
Solution II: Customer provides the cell line, drugs and probes (includes the User Manual). Crnbio follows the protocols described above, or the customer/( the third part) provides the protocols.
2. Pathological Biopsy for ISH
Solution I: Customer provides the pathological biopsy and probes (includes the User Manual). Crnbio follows the protocols described above.
Solution II: Customer provides the pathological tissues and probes (includes the User Manual). Crnbio follows the protocols described above, or the customer/( the third part) provides the protocols.
Service Quote (Customer provide the probes and the samples)
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