1. Use embryos in the oocyst stage at 8-10 days after fertilization for salmonids and at 1 day (20°C) for goldfish and loach.
2. The fertilized egg is placed in Ringer.
3. Egg membrane removal: For salmon, after confirming the position of the embryo with transmitted light, pinch the upper egg membrane of the embryo with tweezers and pinch the wrinkles on the side with another tweezers. The first egg membrane is formed, and the wrinkles formed by the second tweezers are pinched next to each other. Alternately shift both tweezers to make holes, then gently pull them out when the holes are drilled to form a tear. When a tear is formed, shift the position of the tweezers to the end of the tear and pull to both sides to spread the tear. This is repeated to remove the egg membrane.
4. Egg Membrane Removal: For goldfish, immerse in Ringer's solution (pH 8) containing 0.1% trypsin, 0.4% urea and allow to stand for 1-2 hours. When the egg membrane melts, the embryos are collected.
5. Removal of egg yolk from the embryo: In the case of salmon trout, oil spheres are densely packed in the lower part of the embryo. Remove this part from the oil sphere and transfer it to another Ringer's solution. Then gently remove the oil ball with tweezers to make the embryo only.
6. Removal of yolk from the embryo: In the case of goldfish, pulling the membrane of the yolk sac cell into both sides with two tweezers and breaking it, the yolk cells in the embryo jump out without breaking. Only yolk cells are drawn and the remaining embryos are transferred to Ringer in another Petri dish.
② Embryo processing
1. The removed embryo is placed in 0.075M KCl solution and subjected to hypotonic solution treatment.
2.Observe the embryos carefully from 10 minutes after starting the treatment and transfer them to Carnois's solution (methanol: acetic acid = 3:1) using a pipette from embryos that have expanded. Transfer carefully or the cells will be scattered. Pipettes that have been in contact with Carnoy's solution should not be returned directly to the hypotonic solution! Wash the Carnoa's solution in a separate container before returning it to the hypotonic solution.
3. After all embryos have been transferred to Mr. Carnoah's solution, the embryos are transferred to a fixed bottle containing a new Dr. Carnoah's solution. Let the bottle shake slowly.
4. After 10-20 minutes, discard Carnoa's solution and add new Carnoa's solution.
5. This shall be stored in a stocker at-30℃ and chromosome samples shall be prepared as appropriate.
③ Creating a Preparat
Cleaning the Slide Glass
Use clean slides for preparing chromosome specimens. If possible, wash with distilled water by ultrasonic cleaning and keep in ethanol. (takes some time)
Preparation of chromosome specimens（Chopping法）
1.-Embryos stored from a stocker at 30℃ are produced.
2. A drop of DW is placed on a slide with an injection needle, and one embryo is placed on it.
3. Tap the embryo with a knife blade and make it finer until it becomes milky white. When the cell fluid has dried, add a small amount of DW.
4. When the cells are finer, add Ms. Carnoa's solution with a pipette and spread the cells on a glass slide.
5. Ignite Mr. Carnoah's solution and dry the cells so that there is no fire around it.
6. Acetic acid remains on the slide and is discarded and placed on a mappe.
7. Put it on the slide box to prevent dust from sticking.
Preparation of chromosome specimens (Air Dry method)
1.-Put out embryos stored from a stocker at 30℃.
2. Place the slide on a wet paper towel.
3. Place one embryo in a 1.5 ml test tube and add Carnoa's solution to the tube at a concentration of about 200 μl.
4. Embryos are crushed with a yellow tip attached to a pipetman, and the cells are taken in and out to form a cell suspension.
5. Place the slide on a wet paper towel.
6. Blow your breath on the slide and make cloudy.
7. Immediately drop the cell suspension from about 30cm above the glass slide.
8. Drop a few more drops so that they do not overlap.
9. Move the sliding glass to the mapper and let it dry.
10. Put it in a box to prevent dust from sticking.
1. Put half the distilled water (PBS is also acceptable) in the stained bottle.
2. Add 5 ml of Giemsa's solution and mix well.
3. Place slides with chromosome specimens in baskets (for Giemsa).
4. The basket is soaked in Giemsa's solution and stained for 5 to 10 minutes.
5. Remove the slides from the basket and wash with distilled water.
6. Place the front and back side of the Case on a mapper to make sure that there is no mistake and dry it.
7. Store dust-free and observe as soon as possible.
1. Set a slide glass with a chromatographic specimen on the biological microscope.
2. Review the whole bird's eye and check for metaphase chromosome images.
3. If the chromosome image appears to be frequent, observe it evenly from the edge and take a photograph if there is a good sample.
⑤ Preparation of nuclear plate specimens
1. Count the number of chromosomes in the photo and check for expected numbers or overlapping chromosomes.
2. Copy each chromosome from the image and line it up on the pedestal.
3. From the position of the centromere, it is divided into the middle yarn type, the next end yarn type, and the end yarn type.
4. Arrange in order from largest to smallest.
1. Transfer a small piece of sample (just a little better) to a 1.5 ml test tube from. When the sample volume is large, the peak becomes difficult to understand at the time of measurement.
2. Place 150 μl of the pre-formulated solution A (stored under refrigeration) in the tube and vortex. Fluid A serves to make cells loose.
3. Vortex for 30 minutes.
4. Turn on the flow cytometer. To turn on the power, first turn on ① the breaker of the transformer, ② the printer (far right), and after 30 minutes, first turn on the pushbutton switch on the upper side of the switch on the rear side of the main body. Then, turn on the rocker switch (a switch that moves like a seesaw) at the bottom of the push-button switch. The reverse is true when it is deleted.
5. Fill in the lighting time of the fluorescent lamp.
6. Add 600-800 μl of solution B (DAPI) to the tube containing the sample and continue vortexing. DAPI: DNA-fluorescent stain
7. Fill the sample from above the tube with the mesh filter and drop it through the mesh. Add 200 μl of Solution B from the top of the mesh, add 200 μl of DW, and vortex thoroughly. Pressure may be applied from above the rubber cap filter prior to vortexing. (Go down, especially without applying pressure)
8. Press the CLEAN button on the flow cytometer to clean the flow path.
9. Wear the sample (the first control) and push the measurement in STOP→START order.
10. When a wavelength appears on the screen, adjust the peak of the wave to the value of 100. Press the GAINOOCUP or DOWN key to adjust (roughly 485 is exactly right) →ENTER or CLEAR.
11. If there is too much sample, press the Adjust →ENTER with SPEEDOOCUP or DOWN. If the peak is broad (the width of the bottom side is wide), dilute with solution B +DW=1:1.
12. Print the measurement results. STOP→FUNC→PRINT
13. Remove the sample and press the CLEAN to clean the flow path. The sample inlet is immersed in the sample, so the sample is washed by applying DW.
14. Samples are measured (repeats of 9, 11-13). If you want to compare multiple samples together, mix the samples. When the sample solution is low, add solution B and DW.
15. Turn off the power when the measurement is finished.
16. Write down the usage time of the fluorescent lamp in the usage book.
17. Cleaning the mesh: Let it soak in water and remove any dirt from the water stream. Clean with DW.