[0036] Example 1 Detection of the number of transgenic events
[0037] Plant softening treatment
[0038] 1. Transfer the sample to a petri dish and dry it in a constant temperature oven at 37°C for 48h
[0039] 2. The sample is placed in the tissue grinder, and the tissue powder is collected after sufficient grinding
[0040] 3. Take 1g of powder sample, mix well, transfer it into EP tube and wrap the tissue powder with 3% agar, and put it into the agar after it cools and solidifies.
[0041] Fix in FAA fixative solution (50% concentration for young plants and 70% concentration for old hard tissues) for 24h.
[0042] 4. Take out running water and rinse for 2-4h
[0043] 5. For long-term storage, it can be transferred back to FAA fixative
[0044] Paraffin-embedded sections of samples 1. Materials: fresh tissues were fixed in FAA fixative for more than 24 hours. Take the tissue out of the fixative solution, trim the tissue at the target site with a scalpel in a fume hood, and place the trimmed tissue and the corresponding label in a dehydration box.
[0045] 2. Dehydration: Put the dehydration box into the hanging basket and dehydrate with gradient alcohol in sequence in the dehydrator. 75% alcohol 4h-85% alcohol 2h-90% alcohol 2h-95% alcohol 1h- absolute ethanol I 30min- absolute ethanol II 30min- alcohol benzene 5-10min- xylene I 5-10min- xylene II 5- 10min-wax I1h-wax II 1h-wax III 1h.
[0046]3. Embedding: The tissue soaked in wax is embedded in an embedding machine. First put the melted wax into the embedding frame, and before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool at -20℃, after the wax solidifies, remove the wax block from the embedding frame and trim the wax block.
[0047] 4. Slicing: Place the trimmed wax block on a paraffin microtome to slice, with a slice thickness of 4-6 μm. Floating the slices Flatten the tissue on warm water at 40°C in a spreader, pick up the tissue with a glass slide, and place it in a 60°C oven to bake the slices. After the water is dried and the wax is baked, take it out and store it at room temperature for later use.
[0048] Dewaxed paraffin sections to water
[0049] Put the slices into xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and distilled water for washing.
[0050] antigen retrieval
[0051] Tissue sections were placed in a retrieval box filled with EDTA antigen retrieval buffer (pH 9.0) for antigen retrieval in a microwave oven. After medium heat to boiling, switch off the power at an interval of 10 minutes from medium to low heat to boiling. During this process, the buffer should be prevented from over-evaporating, and the tablets should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time.
[0052] Immunofluorescence staining
[0053] Add fluorescently labeled antibodies (cy3-bar antibody and FITC-EPSPS antibody): Gently shake off the blocking solution, drop the primary antibody prepared in PBS in a certain proportion on the slices, and incubate the slices in a humid box at 4°C overnight. (Add a small amount of water to the wet box to prevent the antibody from evaporating);
[0054] DAPI counterstained nuclei: the slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time. After the sections were slightly dried, DAPI staining solution was added dropwise to the circle, and incubated at room temperature for 10 minutes in the dark;
[0055] Mounting: The slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time. After drying, the sections were mounted with anti-fluorescence quenching mounting medium.
[0056] Microscope photo
[0057] Sections were observed under a Nikon inverted fluorescence microscope and images were collected.
[0058] Result analysis
[0059] like figure 1.A green observed for PAT/bar protein, blue fluorescence of B cell nuclei (background fluorescence), C superposition of blue and green. If the sample observes as figure 1 As shown in C, it means that there is only PAT/bar protein in the sample, which means that the sample contains a transformation event.
[0060] figure 2.A Red observed for CP4-EPSPS protein, blue fluorescence of B cell nuclei (background fluorescence), C superposition of blue and red. If the sample observes as figure 2 As shown in C, it means that there is only CP4-EPSPS protein in the sample, which means that the sample contains a transformation event.
[0061] image 3.A green observed for PAT/bar protein, B red observed for CP4-EPSPS protein, C blue fluorescence of nuclei (background fluorescence), superposition of blue, red and green. If the sample observes as image 3 As shown in C, the sample contains two transformation events.
