Integral in-situ hybridization method for sepiella maindroni embryo
A technology of needleless cuttlefish and in situ hybridization, which is applied in the direction of biochemical equipment and methods, and the measurement/inspection of microorganisms, can solve problems such as the decline of cuttlefish resources, reduce the time required for analysis, have fewer experimental steps, and improve The effect of hybridization signals
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Embodiment 1
[0022] The method for whole body in situ hybridization of squid squid embryos, including tissue rehydration, permeabilization, fixation, prehybridization and hybridization, washing after hybridization, blocking, antibody, and color development, wherein the permeabilization treatment steps are: rehydrating The membrane-removed embryos were placed in a solution containing proteinase K and salicylic acid, and the embryos were digested. The egg membrane of the needleless cuttlefish is thicker, and there are pigments in the embryo, so the transparency is poor, and it is not easy for the probe to penetrate and develop color. It is necessary to manually remove the membrane and permeabilize the embryo during the experiment. Proteinase K has the ability to digest The function of surrounding the target DNA protein can make the probe easier to enter the cells of the embryo, increase the chance of the probe binding to the target nucleic acid, and improve the hybridization signal, but the c...
Embodiment 2
[0036] The method for whole-body in situ hybridization of squid squid embryos, the specific steps are as follows:
[0037] A) Tissue rehydration: place the embryos after demembrane removal in 75%, 50%, and 25% methanol solutions for gradient rehydration for 5 minutes each, and then wash with 1×PBST solution 3 times, 5 minutes each time;
[0038] B) Permeabilization treatment: place the rehydrated embryos in a solution containing 10 µg / ml proteinase K and 1.2 µg / ml salicylic acid, digest the embryos at 37°C, and digest the embryos for ≤24 hours for 8 minutes. Embryos >24 hours digested for 22 minutes;
[0039] C) Fixation: immerse the permeabilized embryos in 4% PFA-PBS solution containing 0.01% threonine, fix at room temperature for 15 minutes, and then wash with 1×PBST solution for 3 times, 5 minutes each time. The ratio of L-threonine and D-threonine is 100:3.5;
[0040] D) Pre-hybridization and hybridization: Add 1ml of pre-hybridization solution dropwise to the fixed emb...
Embodiment 3
[0046] The method for whole-body in situ hybridization of squid squid embryos, the specific steps are as follows:
[0047] A) Tissue rehydration: Place demembraned embryos in 75%, 50%, and 25% methanol solutions for gradient rehydration for 5 minutes each, and then wash with 1×PBST solution 3 times, 5 minutes each time;
[0048] B) Permeabilization treatment: place the rehydrated embryos in a solution containing 10 µg / ml proteinase K and 1.2 µg / ml salicylic acid, digest the embryos at 37°C, and digest the embryos for ≤24 hours for 10 minutes. Embryos >24 hours digested for 30 minutes;
[0049] C) Fixation: immerse the permeabilized embryos in 4% PFA-PBS solution containing 0.01% threonine, fix at room temperature for 15 minutes, and then wash with 1×PBST solution for 3 times, 5 minutes each time. The ratio of L-threonine and D-threonine is 100:4.2;
[0050] D) Pre-hybridization and hybridization: Add 1ml of pre-hybridization solution dropwise to the fixed embryos, pre-hybrid...
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