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Protocols

Plasmid part

PCR

The experiment uses a KOD -Plus- PCR kit. Which includes the following:

KOD -Plus- (1.0U/µL) polymerase

dNTPs

MgSO4

Buffer for KOD -Plus-

1.Preparation of PCR reaction solution
Add the following into the EP tube
a.33 µL of distilled water.
b.5 µL of 10X buffer (1x)
c.5 µL of 2nM dNTPs (0.2each)
d.2 µL of MgSO4
e.1 µL of 10 mpol/µL Prime #1
f.1 µL of 10 mpol/µL Prime #2
g.1 µL APP 695 DNA template
h.1 µL KOD-Plus2.

2. Set up PCR program
a.Pre-denaturation: 94 ˚C, 2 min
b.Denaturation: 94 ˚C, 15 sec
c.Annealing: (Tem-5) ˚C, 30 sec
d.Extension: 68 ˚C, 1 min/kb
(Repeat the above b-d steps for 35 cycles)

Electrophoresis

1. Making the Gel
1.Apply 0.6g Agarose with 60 ml of Agarose1×TAE in the beaker
2.Put the beaker in Microwave for 45 sec
3.Apply 6 µl of Yeasen Dye
4.Cool the solution in Gel Staining box for 20 min

2.: Electrophoresis gel
5.Add Purple loading dye (6x) (biolabs) into DNA sample * 10µl
6.Apply DNA sample in Gel Wells * 50µl
7.Set the machine on 120 voltage for 40 min.

DNA Gel Extraction

1.Put DNA under UV light, use a scalpel to cut the DNA fragment.
2.Transfer the gel into micro centrifuge tube
3.Weight the DNA gel = 0.2 g
4.Add 600 µl of Buffer DE-A, heat the tube to 75 ˚C until the gel is completely dissolved.
5.Add 300 µl of Buffer DE-B
6.Place a Miniprep Column into a 2ml microfuge tube. Transfer the solution agarose into the column. Centrifuge at 12000 × g, *1MIN
7.Discard the filtrate from the microfuge tube, add another 500 µl of Buffer W1 into the column. Centrifuge at 12000 × g, *30 SEC
8.Discard the filtrate from the microfuge tube, add 700 µl of Buffer W2. Centrifuge at 12000 × g, * 30 SEC
9.Perform the previous step over again with caution, this is to ensure the complete removal of salt
10.Discard the filtrate, centrifuge at 12000 × g, * 1MIN
11.Transfer the miniprep column into 1.5 ml microfuge tube
12.Add 25 µl of Eluent to the center of the membrane, put in room temperature for 1 min, then centrifuge at 12000 × g, * 1MIN

Enzyme digestion

1. Add the following solution to the EP tube
a.Enzyme 1 *1 µl (for site 1)
b.Enzyme 2 *1 µl (for site 2)
c.10 ×buffer 2 µl
d.Target DNA sequence 16 µl
2. Add the following to another EP tube
e.Enzyme 1 *1 µl (for site 1)
f.Enzyme 2 * 1µl(for site 2)
g.10×Buffer 2 µl
h.Template Plasmid: 0.5 µl/µg
3. Put the two EP tubes in the Heating Incubator, set the TEMP to 37 ˚C for 2 hours.

Plasmid construction

1.The following purification step is done on both DNA and plasmid template.
a.Take 20 µl of both DNA and Plasmid
b.Add 60 µl of Buffer DE-A, heat the tube to 75 ˚C until the gel is completely dissolved.
c.Add 30 µl of Buffer DE-B
d.Place a Miniprep Column into a 2ml microfuge tube. Transfer the solution into the column. Centrifuge at 12000 × g, *1MIN
e.Discard the filtrate from the microfuge tube, add another 500 µl of Buffer W1 into the column. Centrifuge at 12000 × g, *30 SEC
f.Discard the filtrate from the microfuge tube, add 700 µl of Buffer W2. Centrifuge at 12000 × g, * 30 SEC
g.Perform the previous step over again with caution, this is to ensure the complete removal of salt
h.Discard the filtrate, centrifuge at 12000 × g, * 1MIN
i.Transfer the miniprep column into 1.5 ml microfuge tube
j.Add 25 µl of Eluent to the center of the membrane, put in room temperature for 1 min, then centrifuge at 12000 × g, * 1MIN
2.Take 14 µl of DNA template and 3 µl of Plasmid template
3.Add 2 µl of 10 × buffer
4.Add 1 µl of T4 DNA ligase
5.Put the solution in a 17 ˚C incubator overnight for 16 hours.

E.coli transformation

Part 1: implant plasmid
1.Take 100 µl of Competent Cell (E.Coli) in two different ep tube, stocked in -80 ˚C. Put it in Ice Bath.
2.Add 0.5 µl of recombinant plasmid into each competent cell tube. Stock in ice bath for 30 min
3.Put tube in 42˚C water bath for 90 sec, then quickly put back to Ice Bath*2-3MIN
4.In each tube, add 900 µl of sterile LB culture medium, mix the solution
5.Put the put in a 37˚C shaker for 45 min at 150 rpm.
Part 2: Bacteria grow and reproduce
1.Take 100 µl of the cell from each tube after shaking.
2.Spread the cell evenly on the agar medium with a sterile spreading rod. (the agar medium is the same as the previous LB medium with antibiotics)
3.Leave the two plate upside down in 37˚C incubator overnight (16h)

Western blot

1.SDS-PAGE
1）Making separating gel: 15ml
a.4.0 ml of distilled water
b.5.0 ml of 30% Acr-Bis (29:1)
c.5.7 ml of 1M Tris, pH8.8
d.0.15 ml of 10%SFS
e.0.15 ml of 10% Ammonium persulphate solution
f.0.009 ml of TEMED
2）Making Stacking gel: 4 ml
a.2.7 ml of distilled water
a.0.67 ml of 30% Acr-Bis (29:1)
b.0.5 ml of 1M Tris, pH 6.8
c.0.04 ml of 10% SDS
d.0.04 ml of 10% ammonium persulphate solution
e.0.004 ml of TEMED
3）Wait 40 min for Gel to cool down.
4）Heat 15 µl sample in 100˚C for 10 min, then put heated sample in centrifuge 12000 rpm* 1min
5）Insert the vertical electrophoresis gel machine properly.
6）Inject 15 µl sample, 10 µl Blue plus protein marker （10kd-180kd）
7）Run at 80 voltage for 1 hour on stacking gel, 120 voltage for 1 hour on separating gel
2.During SDS-PAGE electrophoresis, prepare the membrane transfer buffer in advance and place it in the 4 ˚C to enhance the membrane transfer efficiency. After electrophoresis, washing gel with Milli-Q ultrapure water to remove swimming fluid on the gel surface. At the same time, PVDF membrane was immersed in methanol for activation. Then, PVDF and gel were placed in the pre cooling fluid to balance for about 10 min to improve the efficiency of the film transfer.
3.Wet transfer: making the film transfer device commonly known as “sandwich structure”, during the period, the bubbles were crushed by centrifuge tube. The film transfer device consists of (from bottom to top) black cathode plate, 3mm filter paper, gel, PVDF film, 3mm filter paper and white anode plate.Then, add frozen ice bags on both sides of the membrane transfer tank and transfer the membrane at a constant current of 300mA for 1 h (generally, the membrane transfer time of 20-100kd protein is 1 h). After membrane transfer, clean PVDF membrane with milli-q ultrapure water twice.
4.Blocking, hybridization and development: block with 5% skimmed milk or 5% BSA with TBST buffer solution and place it on the shaking table at room temperature for 1 h. Then rinse three times with TBST buffer solution for 5 min each time. Prepare primary antibody diluent with 5% BSA, incubate the cut PVDF membrane with primary antibody, and shake in a 4˚C overnight. The next day, rinse 3 times with TBST buffer for 10 min each time. Then add the secondary antibody, incubate at room temperature for 1 h, wash the membrane with TBST buffer solution for 10 min. Prepare ECL substrate reaction solution and develop and analyze it with GE ImageQuant LAS4000.

Drosophila part

Preparation of drosophila culture medium

The food of the experimental drosophila is made with brown sugar-cornmeal-yeast medium, as shown in table.

1. Conventional drosophila culture medium
Specific methods:
1) Pour 5L of water, all brown sugar and agar together into the rice cooker and heat.
2) Dissolving the cornmeal with 1L of water and stir well.
3) When the water boils in the rice cooker, pour the dissolved cornflour paste into the rice cooker and heat it (in and after the heating process pay attention to stirring, prevent sticky pot).
4) Take an additional 600ml warm water to dissolve the yeast.
5) After the previous "brown sugar corn porridge" is heated to boil, turn off the power to the rice cooker. Wait for its temperature to be slightly
After reducing (about 60 degrees Celsius), pour the yeast solution evenly in and stir well.
6) Add the preservative (24ml acrylic) and stir well. At this point, the media configuration is complete.
7) Then, pour the matching medium into the culture tube while hot, and plug the cotton plug, place in a cool place to spare.

Chloroquine drosophila culture medium

Specific methods:
1) Pour 5L of water, all brown sugar and agar together into the rice cooker and heat.
2) Dissolving the cornmeal with 1L of water and stir well.
3) When the water boils in the rice cooker, pour the dissolved cornflour paste into the rice cooker and heat it (in and after the heating process pay attention to stirring, prevent sticky pot).
4) Take an additional 600ml warm water to dissolve the yeast.
5) After the previous "brown sugar corn porridge" is heated to boil, turn off the power to the rice cooker. Wait for its temperature to be slightly
After reducing (about 60 degrees Celsius), pour the yeast solution evenly in and stir well.
6)Add the preservative (24ml acrylic) and stir well. At this point, the media configuration is complete.
7) 25 tubes of two different concentrations of drug solutions were prepared. 2mg/mL chloroquine solution (25mL water +0.5g chloroquine) and 5mg/mL chloroquine solution (25mL water +1.25g chloroquine). Mix the solution in a scroll and then shake it in a shaker.
8) Add solution into prepared culture medium.
9) Then, pour the matching medium into the culture tube while hot, and plug the cotton plug, place in a cool place to spare.

Courtship choice assay

Preparation：
1)Hybridization：
⁃Virgin fru-Gal4 × male W1118
⁃Virgin fru-Gal4 × male UAS-Dcr2
⁃Virgin fru-Gal4 × male UAS-APP
2)Culturing at a constant temperature of 23.5℃. After feathhacthingering, transfer the temperature to 25℃.
3)Alternative drosophila：
⁃Naive maleMale drosophila that are cultured for 3 days after being judged as naive by meconium are cultured individually in EP tubes with perforation at the top for oxygen.
⁃Young females are OR red-eyed virgin for 3 days and old females are w1118 virgin for 30 days (White and red eyes are designed to distinguish young and old female drosophila)
⁃The experimental naive was collected with the virgin when the old virgin are 27 days old.

Experimental process：
1)Carbon dioxide is first passed into the test tube to anaesthetize drosophila in it. Then, one naive, two young virgin, and two older virgin are taken.
2)Place the drosophila on a plate that is connected to carbon dioxide and quickly confirm under a microscope that sex, eye color, survival, and wings are normal, as residual or wing-spreading disorders affect the observation of courtship behavior (winging).
3)Be careful not to anaesthetize for too long to prevent drosophila from dying or decreasing activity, affecting courtship behavior.
4)Place the drosophila in a hole of the glass dish used for observation and wait for it to wake up.
5)After all five drosophila were awakened, record all activities for 10 minutes using a shooting instrument.
6)If the female and male drosophila mate successfully within 10 minutes of the experimental process, the data in this group are invalidated because the female that mated successfully have courtship rejection behavior, which would affect the accuracy of the experimental data, and if the drosophila die or the male’s wing vibration are disturbed, the data can also be invalidated.
7)Drosophila used in experiments are discarded after recording and are not reused even if mating is not successful to ensure the accuracy of experimental data.

Data statistics and processing：
1）Importing video into the computer for data statistics.
2）The statistical process is blind: the statistician does not know the genotype of the drosophila in the video to eliminate the effect of psychological expectations (researcher bias) on data collection.
3）The vibration of the male’s one-sided wings is regarded as courtship behavior, and the time spends by the male courting young and old female is counted in the 10-minute test time.
⁃The time when the young female are courted is counted as Tyoung, and the time when the old female are courted is counted as Told.
4）Calculating the courtship index of the male to the female.（Courtship Index，CI）
⁃CIy represents the male's courtship index for the young female, and CIo represents the courtship index for the old female.
⁃CIy = Tyoung/10min x 100%，CIo = Told/10min x 100%
5）Calculate the courtship preference index of the male.（Preference Index，PI）
⁃In order to better identify the male's tendency to choose the age of the courtship female, we defined the male's courtship preference index by reference to the previous literature.（Preference Index，PI）
⁃PI=(CIy−CIo)/(CIy+CIo)×100%
⁃PI>0 represents a preference for young female, PI<0 represents a preference for old female, and PI=0 represents an equal preference for young and old female.

Pupation height

Preparation：
1）Hybridization
Virgin APPL-Gal4 × male W1118
Virgin APPL-Gal4 × male UAS-Dcr2
Virgin APPL-Gal4 × male UAS-APP
2）Incubator: constant temperature 25℃, constant humidity 60-70%, day and night alternate (cycle: 12 hours day and 12 hours night), keep oxygen sufficient.
Data statistics and processing：
1）Fruit flies aged 7-8 days after hybridization were selected to ensure that in the pupal stage, culture tubes of corresponding genotypes of fruit flies were selected and placed side by side in a certain order in front of a white background to facilitate the subsequent determination of genotypes. Parallel photos were taken aiming at the frame and imported into Photoshop.
2）Base line: Half of the surface of the medium, the horizontal diameter.
3）The pupa height on the surface of the culture medium was 0, and the pupa number on the surface was counted under the microscope and counted separately.
4）Select the most upright test tube, use the ruler tool to measure its pixel height and diameter, open the measurement record window to set the custom measurement ratio, and save the preset.
⁃The height pixel length corresponds to 99mm logical length, and the diameter pixel length corresponds to 25mm logical length.
5）Hold shift and pull the scale vertically down from the middle of each pupa to the middle of the medium. Measure the length of the pupa using the preset height scale and diameter scale respectively, excluding pupa on the medium surface. Note that the number of pupa per genotype does not exceed 100.
6) Import the measured data of height scale and diameter scale into Excel respectively, and calculate the average value of the two scales to increase data accuracy.
7) Multiple people test the same tube and take the average as the final result to increase the accuracy of data.

Adult locomotor test

Preparation：
1）Hybridization
Virgin APPL-Gal4 × male W1118
Virgin APPL-Gal4 × male UAS-Dcr2
Virgin APPL-Gal4 × male UAS-APP
2）Incubator: Before drosophila become adults, the temperature is kept at 17℃ (in order to suppress the phenotype of drosophila's wing spreading disorder, so that drosophila has the ability to climb tubes). After drosophila become adults, the temperature is kept at 29°, and the humidity is 60-70%. The day and night are alternating (period: 12 hours of day and 12 hours of night), and oxygen is sufficient.
Experimental process：
1) When hybrid offspring become adult and have 2 days of age, transferring drosohpila in the tube to the vibration table (pay attention to the transfer of drosophila to the bottom of the tube, and quickly connect the tube with the mouth of the shaking table. Drosophila are herded inside the shaker tube and the top of the shaker tube is covered with small holes).
2) Fix the pipe on the shaking table in the previous sequence and tighten the top screw to fix the pipe to prevent it from falling off during vibration.
3) After setting up the camera equipment, turn on the shaking table and start recording. A total of five images before and after the shaking are recorded.
Data statistics and processing：
1) The height at the bottom of the vibrating table tube is 0, and the number of pupae on the surface is counted in the video for separate statistics.
2) Select the most upright test tube, use the ruler tool to measure its pixel height and diameter, open the measurement record window to set the custom measurement ratio, and save the preset.
-The pixel length of height corresponds to 99mm logical length, and the pixel length of diameter corresponds to 25mm logical length.
3) Hold shift, pull the scale vertically down from the middle of each pupa to the middle of the medium, and measure the length according to the preset height scale and diameter scale respectively, excluding pupae on the medium surface, and note that the number of pupae for each genotype does not exceed 100.
4) Import the measured data of height scale and diameter scale into Excel respectively, and calculate the average value of the two scales to increase data accuracy.
5) Multiple people test the same tube and take the average as the final result to increase the accuracy of data.

Drosophila learning and memory experimenttest

Preparation：
1) Hybridization：
Virgin elav-Gal4 × male W1118
Virgin elav-Gal4 × male UAS-Dcr2
Virgin leave-Gal4 × male UAS-APP
2）Culturing at a constant temperature of 23.5℃. After feathering, transfer the temperature to 25℃.
3）Alternative drosophila：
-Naive (bred for 3 days) are cultured in EP tubes (perforated to prevent the flies from suffocating).
-Virgin : W1118 3days. On the second night, they mate with W1118 male. On the third day, female with successful mating (whether their tails are connected with eggs) are selected for the experiment.

Experimental process：
1）Carbon dioxide is pumped into the EP tube to stun the drosophila inside. They are placed on a carbon-dioxide-coated board with confirming female flies mated successfully. The sex, survival, and normal wings were quickly determined under a microscope (the observation of broken wings or flapping wings can affect courtship behavior).
2）* Be careful not to anesthetize for too long to prevent drosophila from dying or being less active and affecting courtship behavior.
3）Place the drosophila in a hole of an observation dish and wait for it to wake up.
4）After both drosophila wake up, they are left alone for 10 minutes to familiarize themselves with each other and the environment. A camera is used to record their movements for 10 minutes.
5）After 10 minutes of recording, drosophila are left standing for 50 minutes, giving the males time to learn and remember. (Because mated females reject offers from other males so frequently that within 50 minutes, male learn that the female can't mate again.) The camera is then used to record all activity for 10 minutes after standing.
6）Drosophila are used as waste treatment after recording to ensure the accuracy of experimental data.

Data statistics and processing：
1) Importing video into the computer for data statistics.
-The statistical process is blind: the statistician does not know the genotype of the drosophila in the video to eliminate the effect of psychological expectations (researcher bias) on data collection.
2) The unilateral wing vibration of male is regarded as courtship behavior, and the courtship time of male to mated female is calculated in the two 10-minute test periods.
⁃The courtship time within the first ten minutes is considered Tinicial, and that after ten minutes is considered Tfinal.
3）Calculating the courtship index of the male to the female.（Courtship Index，CI）
4）CIi represents the courtship index of male in the first ten minutes, and CIf represents the courtship index in the last ten minutes.
5）CIi = Tinicial/10min x 100%，CIf = Tfinal/10min x 100%
6)Calculating the learning index of male（Learning Index，LI）
In order to better identify the Learning and memory ability of male, we define the Learning index (LI) of male by referring to previous literature.
⁃LI = (CIi-CIf)/CIi x 100%
LI=1 indicates that the male have strong learning and memory ability. Conversely, LI=0 means that males have little ability to learn and memorize.

Wing expansion defect test

Preparation：
1)Hybridization：
Virgin APPL-Gal4 × male W1118
Virgin APPL-Gal4 × male UAS-Dcr2
Virgin APPL-Gal4 × male UAS-APP
2）Culture at a constant temperature of 25℃.
Experimental process：
Offspring of the hybrid drosophila are taken and placed in an EP tube and frozen in a refrigerator at -80 ℃ for 15minutes. They are then removed and placed in a small dish with AGAR and photographed under a microscope.

Anatomy of drosophila larva/AO dyeing

Preparation:
1） Hybridization
For VNC
Virgin APPL-Gal4 × male W1118
Virgin APPL-Gal4 × male UAS-Dcr2
Virgin APPL-Gal4 × male UAS-APP
For eye disc
Virgin GMR-Gal4 * male W1118
Virgin GMR-Gal4* male UAS-Dcr2
Virgin GMR-Gal4* male UAS-APP
2）Incubator: constant temperature of 25℃, humidity 60-70%, alternating day and night (period: 12 hours of day and 12 hours of night). Remember to keep adequate oxygen.
Experimental process：
1.Rough anatomy:
1)Fold the tissue several times and clip it to the side of the microscope. Then adjust the lighting of the microscope to top-down lighting.
2)Add a certain amount of 0.1% PBST with 0.1% concentration to a small black-bottom disk to ensure that the liquid completely reaches the bottom of the disk.
3)If there are still worms in the culture tube, use an air gun to inject carbon dioxide gas to anesthetize them, and pour them into the treatment tank.
4)Take the filial of the above hybrids and cultivate them until they grow into the third instar larvae , which are fat and still on the tube wall and no longer peristaltic. Roll the larvae several times on the tube wall until the medium it touched has been removed and put them into a small disk.
5)Adjust the microscope spiral until the insect body is clearly seen, and judge the front and back ends (the end with black mouthparts and large movements is the front end), and place the front end to the left and the back end to the right.
6)Hold the thick tweezers in the left hand to clamp the 1/3 to 1/2 of the front part of the insect body, and use the fine forceps in right hand to cut it down. The separated tail part of the insect body is removed with tweezers, and wipe off the first part on the prepared tissue. Clamp the head tissue with the thick tweezers in the right hand, then insert the fine tweezers in the left hand into the mouth part of the larva, and then use the thick tweezers in the right hand to slowly open the epidermis along the fine tweezers, turning the inside and out to expose the internal structure and the epidermis Wrapped on fine tweezers.
7)Remove the tissue from the fine tweezers with coarse tweezers, keeping the skin inside.
8) add 990uL 0.1% PBST into the EP tube and put it into the dissected tissue. Write the genotype and concentration on the cover of the EP tube.
2. Dyeing
1) Add 10uL AO （Acridine Orange）to the organized 990ul EP tube in the previous step (990uL 0.1% PBST with tissue).
2) The EP tube is wrapped with tin foil to avoid light treatment to ensure that AO dyefuel will not quickly quench because of too long exposure to light. The EP tube with tin foil is placed on the rotary table and rotated for 2-3 minutes.
3. Precise anatomy
1) Take out the EP tube, use the needle to suck out the mixture of AO dye and PBST, pour it into the waste liquid tank, add a small amount of PBST solution, shake it a few times by hand, and clean the dye on the tissue surface.
2) Take out the slide and wipe it clean. Place it on the microscope bench with the frosted side of the slide facing up.
3)The tissue with a small amount of PBST was dropped onto the slide with a pipette, and the microscope lighting was switched to bottom-up lighting.
4)Remove fat and other irrelevant tissues with an anatomical needle and put them aside. Dissecting VNC or eye disk as completely as possible.
5)The tissue on the epidermis is stripped clean (as a buffer during capping to prevent VNC or Eye disc from being crushed during dissection) and placed to the left of VNC (Ventral Nerve Cord) or eye disk at a distance from VNC or eye disc to prevent overlap during capping.
6)Fold another tissue so many times that it almost cannot be folded anymore, and use a hard corner to wipe away any irrelevant tissue.
7)Pick up one side of the cover glass with tweezers, contact the liquid with the other side first, and then slowly put it down to reduce bubbles and uniformly cover the liquid under the cover glass.
4. Take photos:
1) Place the finished slide specimen under a photo microscope and adjust the mode to red light.
2)First find the tissue with low magnification, then move the tissue to the center of the field of vision, and then change to high magnification.
3)Adjust the fine foci spiral until the red point is clear.
4)Record VNC or Eye Disk images under different exposures to prevent some luminous points from being too dark or bright to be counted under specific exposures.
5) Set up the corresponding date-genotype-culture temperature folder and put the pictures in.
Data statistics and processing：
1)Import the image into Photoshop.
2)Count the number of glowing red dots: Use the brush tool to put white dots on each glowing point.
3)VNC: the number of glowing points on the stick.
4)Eye disk: first count the brightest line on the shallower disk and the number of luminous points to the bottom, then count the number of luminous points on the whole shallower disk, and finally calculate the percentage of the former in the latter.
5) Import the data into Excel and put them into the corresponding genotypes.

Anatomy of drosophila larva/lyso-tracker dyeing

Preparation：
1）Hybridization
For VNC
Virgin APPL-Gal4 × male W1118
Virgin APPL-Gal4 × male UAS-Dcr2
Virgin APPL-Gal4 × male UAS-APP
For eye disc
Virgin GMR-Gal4 * male W1118
Virgin GMR-Gal4* male UAS-Dcr2
Virgin GMR-Gal4* male UAS-APP
2）Incubator: constant temperature of 25℃, humidity 60-70%, alternating day and night (period: 12 hours of day and 12 hours of night). Remember to keep adequate oxygen.
Experimental process：
1）Rough anatomy：
⁃Tissue is prepared in advance is clipped to one side of the microscope, and the microscope is illuminated from top to bottom.
⁃Add PBST with a certain concentration of 0.1% to a small disk with black background.
⁃Take the filial of the above hybrids and cultivate them until they grow into the third instar larvae , which are fat and still on the tube wall and no longer peristaltic. Roll the larvae several times on the tube wall until the medium it touched has been removed and put them into a small disk.
⁃Adjust the microscope spiral until the insect body is clearly seen, and judge the front and back ends (the end with black mouthparts and large movements is the front end), and place the front end to the left and the back end to the right.
⁃The left hand holds the coarse tweezers to clamp the front part of the insect body about 1/3 to 1/2, the right hand holds the fine tweezers to delimit downward, and the separated tail insect body is removed with tweezers and wiped on the prepared tissue.
⁃The left hand inserts the fine forceps into the oral organ of the larva, and the right hand holds the thick forceps and slowly opens the epidermis to reveal the internal structure.
⁃Remove the tissue from the fine tweezers with thick tweezers, keeping the skin inside.
⁃Add 800-1000 ul 0.1% PBST and dissected tissue into the EP tube, place it into the cut tissue, and write the genotype and concentration on the cover of the EP tube.

2）Dyeing
-Attach the needle tube to the tube wall, suck PBST as much as possible, pour into the waste liquid tank, take care not to shake EP tube, do not touch the tissue.
-Add 20uL lyso-tracker.
-Open the water bath pot to 37 ℃, put the EP tube into the water bath for 5 minutes.

3）Precise anatomy
⁃Take out EP tube, suck out Lyso-tracker with needle tube, pour into waste liquid cylinder, and add a small amount of PBST.
⁃The frosted side of the glass slide is upward, the tissue and a small amount of PBST are transferred to the glass slide with a pipette, and the microscope is switched to bottom-up lighting.
⁃The fat and other unrelated tissues are stripped off and put aside with an anatomical needle. The VNC (top two balls, bottom one rod) or eye disk (attached to the VNC ball or stick) should be dissected as completely as possible, leaving the clean skin (as a buffer for capping) in a clean corner, separated from each other to prevent overlapping during capping.
⁃Fold thea other new piece of tissuepaper several times and wipe away the irrelevant tissue with the harder corner. Pick up one side of the cover glass with tweezers, contact the liquid with the other side first, and then lower it slowly to reduce bubbles and cover the liquid evenly under the cover glass.

4）Take photos：
⁃Put the completed slide specimen under the photo microscope, adjust the mode for red light.
⁃Record VNC or eye disk images under different exposures to prevent some luminescence points from being too dark or too bright to be counted under specific exposures.
⁃Create the corresponding date -genotype- culture temperature folder and put the picture in.
Data statistics and processing：
1) Import the image into Photoshop.
2) Count the number of glowing red dots: Use the brush tool to put white dots on each glowing point.
3) VNC: the number of glowing points on the stick.
4) Eye disk: first count the brightest line on the shallower disk and the number of luminous points to the bottom, then count the number of luminous points on the whole shallower disk, and finally calculate the percentage of the former in the latter.
5) Import the data into Excel and put them into the corresponding genotypes.

Effects of drugs on drosophila growth

Preparation：
1) Hybridization：
W1118 female ×X W1118 male (five females, three males)
2）Incubator: constant temperature of 25℃, humidity 60-70%, alternating day and night (period: 12 hours of day and 12 hours of night), keep adequate oxygen, culture for two days.
Experimental process：
1.Count the number of eggs laid by the female
1)All the drosophila in the tube are rotated every three hours for three times in total. After three times, drosophila in the culture tube are dumped.
2) Record the number of eggs laid in each tube: place the tube under a microscope, adjust the light from up to down, and count the number of eggs laid by female in three hours under a microscope.
2.Counting the number of pupa
1）Take out the culture tube and count the number of pupae on the wall of the tube. Keep following up the increasing number of pupae every day for several days until the number of pupae no longer increases (or there are no larvae in the tube).
2) Record the total number of pupae on the tube wall daily.
3. Counting the number of adults
1）Place the tube in the incubator until adult emerges. Then, remove the tube.
2)First, drosophila are stunned by a carbon dioxide air gun, and then the adults are poured onto the board with carbon dioxide. After counting the number, they are poured into the drosophila treatment tank. The daily increase in adult numbers was followed for several days until there was no increase in adult numbers.
Data statistics and processing：
Pupal formation rate = number of pupae/number of eggs
Emergence rate = number of adults/number of pupae
Pupa formation and emergence time

Effects of drugs on drosophila lifespan

Preparation：
1）Hybridization：
-First hybridization: Female UAS-APP × male X Sp/CyO male
2）Male with CyO phenotype are selected from their filial.
-Second hybridization: filial of first hybridization male (UAS-APP/CyO) male ×X female Appl-GAL4
female
3）Incubator: constant temperature of 25℃, humidity 60-70%, alternating day and night (period: 12 hours of day and 12 hours of night). Remember to keep adequate oxygen.
4）Add 0ml, 2ml, and 5ml of chloroquineagentia to the medium.
5）Alternative drosophila: Curly of Oster
Experimental process:
1）The filial obtained after the second hybridization are cultured separately in a new culture tube, and two genotypes of drosophila can be obtained in the second hybridization, respectively:
-Appl > APP (Normal phenotype of drosophila with Alzheimer's disease)
-Appl-Gal4 /CyO (Common drosophila with CyO phenotype)
2）The test tubes are placed in incubators and the number of drosophila with both phenotypes (genotypes) are counted after several days.
Data statistics and processing：
Survival number of drosophila with genotype Appl > APP/survival number of drosophila with genotype Appl -Gal4/CyO