Abstract
RNA in situ hybridization is a powerful technique but can be time consuming and potentially hazardous. We developed a procedure in which nonisotopic in situ hybridization is performed in Vibratome-sectioned tissues that are not dehydrated or embedded in paraffin or resin, thus avoiding additional pretreatment steps to allow the probes to penetrate the cell structure. Predigestion with proteases was not necessary. Hybridization of sections and immunologic detection were performed in microplates to avoid the use of slide-coating agents. Biotinylated cDNA probes were synthesized by PCR. Synthetic oligonucleotide probes chemically labeled with biotin or digoxigenin were also used. Stained sections were mounted on slides for microscopic observation, and various transcripts were successfully detected using different visualization methods. This method is fast and can be easily implemented in other laboratories because it requires minimal expertise in processing biological samples for microscopy.
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Abbreviations
- AP:
-
alkaline phosphatase
- BCIP:
-
5-bromo-4-chloro-3-indolyl-phosphate
- DIG:
-
digoxigenin
- GS:
-
glutamine synthetase
- NBT:
-
nitroblue tetrazolium chloride
- PBS:
-
phosphate buffer saline
- Pipes:
-
piperazine-N,N′-bis(2-ethanesulfonic acid)
- UTR:
-
untranslated region
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Borlido, J., Pereira, S., Ferreira, R. et al. Simple and fast in situ hybridization. Plant Mol Biol Rep 20, 219–229 (2002). https://doi.org/10.1007/BF02782457
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DOI: https://doi.org/10.1007/BF02782457