Method for in situ hybridization of rat periodontal ligament type I collagen mRNA
Experimental procedure
1. Sample preparation
Eighty male Wistar adult rats weighing 250 ± 20g were selected, and after injecting excessive anesthetics into the rats, the left ventricle was perfused with 4% paraformaldehyde. The mandible was dissected out, and only the bone segment of the molar region was retained, soaked in 4% paraformaldehyde and fixed for 6 hours. Move the specimen into decalcified solution (containing 0.7g / L EDTA, 8mg / L sodium potassium tartrate, 0.14g / L sodium tartrate, 99.2ml / L HCL) and store at 4 ℃ for two weeks. Wash the decalcification solution, dehydrate with gradient ethanol and embed in paraffin. Each mandibular specimen was cut into 3 tissue sections, the thickness of which was uniformly controlled to 5 μm, and mounted on siliconized glass slides.
2. Preparation and labeling of probes
An anti-sense oligonucleotide probe with a length of 39 bases was used and the sequence was: 5'-GCC GTC TTC AGA GCT GTA AAC GTG GAG GCA AGG AGT CCC-3 '. Digoxigenin 11-dUTP was labeled with the digoxin oligonucleotide tailing kit to the 3 'end of the oligonucleotide.
3. In situ hybridization
1) Conventional xylene dewaxing, gradient alcohol to water (Depc water);
2) Soak in 0.2MHC1 for 20 minutes and wash with Depc;
3) Add 50μg / ml proteinase K, incubate at 37 ℃ for 20 minutes, wash with PBS;
4) Soak in 0.2% glycine PBS solution for 20 minutes and wash with PBS;
5) After fixation with 4% paraformaldehyde in PBS for 20 minutes, wash with PBS;
6) Soak in 0.25% acetic anhydride, 0.1M triethanolamine solution for 10 minutes, wash with PBS; alcohol gradient dehydration, dry in air for 1 hour;
7) Prepare hybridization buffer: 0.125mg / ml SSDNA, 0.25mg / ml tRNA, 50% formamide, 12.5% ​​dextran sulfate, dry bath at 104 ℃ for 10 minutes, then naturally cool to room temperature, then add an appropriate amount of DTT, and set aside;
8) Mix the labeled probe with probe diluent to 10μg / ml, and mix with the hybridization buffer at a ratio of 1: 5 to form a hybridization solution;
9) Drop the pre-hybridization solution and incubate at 42 ℃ for 2 hours;
10) Pour off the prehybridization solution, add the hybridization solution dropwise, add a coverslip, and incubate at 42 ° C for 16 hours.
4. Detection of hybridization signals
1) Move the slide to the formamide solution preheated to 37 ° C and shake for 5 minutes until the cover slip comes off;
2) Wash twice with 2 × SSC in a 37 ° C water bath (15 minutes and 30 minutes);
3) Wash twice with 0.2 × SSC in a 47 ° C water bath (15 minutes and 30 minutes) with 1 × maleic acid buffer for 10 minutes at room temperature;
4) Add blocker dropwise, 30 minutes, room temperature;
5) Add freshly prepared 1: 1000 anti-digoxin alkaline phosphatase and incubate at 37 ℃ for 3 hours;
6) After washing with maleic acid buffer, add detection buffer dropwise;
7) NBT / BCIP 1:50 is added dropwise, and the color is developed in the dark at 25 ° C for 13 hours, rinsed with tap water; counterstained with 2% methyl green;
8) The slices are dried, xylene is transparent, and Canadian glue is used for sealing.
5. Controlled experiment
In order to verify the accuracy of the in situ hybridization experiment and the specificity of the results, a series of positive and negative controls including probes, hybridization reactions, and detection system controls need to be set.
1) Probe control: including in situ hybridization with rat skin specimens and in situ hybridization with isometric positive strand probes.
2) Hybridization control: including the elimination of labeled probe hybridization, hybridization with unlabeled probes and hybridization after treatment of specimens with RNase.
3) Comparison of detection system: the step of adding Anti-Dig-AP is omitted during the detection.
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