In situ hybridization is a very informative technique to present mRNA and IncRNA expression patterns of specific genes in tissues. Our purpose in this article is to develop a sensitive method for detecting the specific mRNA localization in coelomic fluid of Sipunculus nudus, which is a crucial fishery resource. We have presented the representative results obtained from our successful experiments by using this method.
It is hoped that our method will be apply to other Sipuncula species for identifying the expression patterns of the specific mRNA and IncRNA in coelomic fluid. Fix the Sipunculus nudus on the dissection table. Open the body of the Sipunculus nudus with small autoclaved scissors.
Then collect and transfer the coelomic fluid with pipette to poly-D-lysine treated microscope slides, spread mildly. Air-dry the slides for an hour at 37 degree Celsius. Wash the slides with DEPC-PBS for three times, five minutes per wash with gentle agitation.
Drain the PBS, add the proteinase K solution on the slides for five minutes at room temperature. Drain the proteinase K solution. Add the PFA solution on the slides for 20 minutes at room temperature.
Drain the PFA solution. Wash the slides with DEPC-PBS for three times, five minutes per wash with gentle agitation. Drain the PBS.
Add 50 microliter riboprobe. Add a coverslip. Put the slides into the wet box and seal well with paraffin film.
Hybridize overnight at 60 degree Celsius. Immerse the slides in the wash buffer and let it stand until the coverslip slides off automatically. Wash the slides with the preheated wash buffer for two times at 65 degree Celsius, 30 minutes per wash with gentle agitation.
Wash the slides with the preheated SSC solution for two times at 65 degree Celsius, 30 minutes per wash with gentle agitation. Wash the slides with the MABT solution for two times at room temperature. 30 minutes per wash with gentle agitation.
Drain the MABT. Add the blocking buffer. Incubate the slides at room temperature for three to four hours.
Drain the blocking buffer. Add the antibody, incubate the slides at four degree Celsius overnight. Drain the antibody solution.
Wash the slides with the MABT solution for four times, 25 minutes per wash with gentle agitation. Drain the MABT. Add the alkaline phosphatase buffer for three times, five minutes per incubation.
Remove the alkaline phosphatase buffer. Add the BCIP-NBT staining solution. Keep the slides in the dark.
The staining time varies from minutes to hours, even over night, which depends on the signal intensity. The staining status should be checked more frequently at the beginning of the staining. For example, with an interval of several minutes, and several hours at the latest of the staining.
The checking time should be as short as it can be to avoid introducing high background signaling due to the exposure to light. The representative signals of in situ hybridization are shown. In situ hybridization of Sipunculus nudus coelomic fluid with anti-sense riboprobe that targets dmrt1 indicates purple staining concentrated in trophoblast cells of the spermatozeugmata, figure 2A and B.Sense riboprobe for dmrt1 did not detect any hybridization signal, figure 2C.
After watching this video, you would have a very good understanding of how to visualize the expression patterns of target mRNA or IncRNA in the coelomic fluid of Sipunculus nudus.
