Preparation method of immunoelectron microscope sample for positioning specific expression protein in cryptosporidium intracellular in different parasitic periods
A cryptosporidium and immuno-electron microscope technology, applied in the field of biology, to achieve the effect of improving the success rate and quality
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Embodiment 1
[0070] 1. Prepare samples
[0071] (1) The cryopreservation tube of HCT-8 cells was thawed by shaking rapidly in a 37°C water bath, and the thawed cells were transferred to 1640 medium containing 2ml of 10% (w / v) FBS, centrifuged at 800rpm for 5 minutes, Discard the old culture medium, add 1ml of 1640 medium containing 10% FBS to resuspend, then add to T25 cell culture flask containing 4ml of 10% FBS 1640 medium and culture until the density grows to 80% HCT -8 cells were passaged, and after two generations of cell passages, cells in good growth state were obtained for subsequent experiments. According to the method of subcultured cells, the resuspended cells were obtained at a rate of 5×10 per well. 5 Cells were seeded into 12-well plates and cultured in 1640 medium with 10% FBS.
[0072] (2) When the HCT-8 cell density grows to 70%-80%, discard the old culture medium, rinse with 1M, PBS of pH=7.3-7.5 for 3 times, add 1640 medium of 2% FBS, and pass through Treat with 0.5%...
Embodiment 2
[0091] 1. Prepare samples
[0092] (1) Thaw the cryopreserved tube of HCT-8 cells in a 37°C water bath by shaking quickly, transfer the thawed cells to 2ml of 1640 medium containing 10% (w / v) FBS, centrifuge at 800rpm for 5 minutes , discard the old culture medium, add 1ml of 1640 medium containing 10% FBS to resuspend, and then add it to a T25 cell culture flask containing 4ml of 10% FBS 1640 medium for culture until the density reaches 80%. HCT-8 cells were passaged, and after two generations of cell passages, cells in good growth state were obtained for subsequent experiments. According to the method of subcultured cells, the resuspended cells were obtained at a rate of 5×10 per well. 5 Cells were seeded into 12-well plates and cultured in 1640 medium with 10% FBS.
[0093] (2) When the HCT-8 cell density grows to 60%-70%, discard the old culture medium, rinse with PBS for 3 times, add 1640 medium with 2% FBS, and treat with 0.5% sodium hypochlorite on ice for 10 minutes ...
Embodiment 3
[0112] (1) Refer to the method of Example 1 to prepare slices, and then perform immunogold labeling: PBS (1M, pH=7.3-7.5) infiltrates slices, room temperature for 20 minutes; concentration 1% BSA to block non-specific protein binding sites, room temperature 20 minutes; 1M PBS containing 1% BSA diluted primary antibody (INS-15 polyclonal antibody, which has been described in the literature "Xu, Guo, Li, et al.Characterization of INS-15, A Metalloprotease Potentially Involved in the Invasion of Cryptosporidium parvum[ J].2019.", the dilution ratio is 1:200) and incubated overnight at 4°C; the samples were rinsed 4 times with 1M, pH=7.3-7.5 PBS containing 0.1% BSA, 5 minutes each time; 1M, pH=7.3-7.5 PBS diluted secondary antibody (gold-labeled goat anti-rabbit, dilution ratio 1:200) and incubated at 37°C for 1 hour; 1M PBS containing 0.1% BSA, pH=7.3-7.5 rinsed sample 4 times, 5 minutes each time; then use ddH 2 O rinse 4 times, 5 minutes each.
[0113] (2) Heavy metal stainin...
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