Western blot protocol (Conlon lab) and Anesthesia, in vitro fertilization, and natural mating of Xenopus tropicalis (Conlon lab): Difference between pages

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imported>Virgilio
(Created page with "Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot] link to protocol document [http://www.unc.edu/~fconlon/Protocols/Wester...")
 
imported>Virgilio
(Created page with "Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot] link to protocol document [http://www.unc.edu/~fconlon/Protocols/TropIV...")
 
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link to protocol document [http://www.unc.edu/~fconlon/Protocols/Western%20and%20lysis.doc]
link to protocol document [http://www.unc.edu/~fconlon/Protocols/TropIVFMating.doc]




Western Protocol
'''Protocol for anaesthesia of adult X.tropicalis'''


(updated 3-17-2011)
Anaesthesia can be used to make the frogs easier to handle during sub-cutaneous injection of hCG, reducing the likelihood of injury to the animals.


:- Place frogs into tanks containing frog system water to a depth of approximately 2 inches.
:- Add ethyl 3-aminobenzoate methanesulfonate salt (Tricaine/MS222; Sigma) to a final concentration of 0.025% (w/v). Tricaine can be stored at 4˚C as a 2.5% stock solution in water. 
:- Allow approximately 30 minutes for the anaesthetic to take effect. Do not leave the frogs in the anaesthetic for longer than is necessary for anaesthesia.
:- Once the procedure is complete, allow the frogs to recover from anaesthesia in shallow water (i.e. not more than 2 inches deep). 


FOR EMBRYOS:


Lysis Buffer (10 ml)
'''Protocol for in vitro fertilization of Xenopus tropicalis '''


{|border="1" cellpadding="5"
This method should routinely result in >70% fertilization per batch of eggs. However, not all males are equally fertile, so two males should be primed per experiment to allow for a backup male should the first be infertile. Four females should allow selection of the best quality eggs and provide enough for several rounds of injections etc.  
|-
! scope="row" style="background:#efefef;" |Lysis Buffer (10 ml)
|500ul 1M Tris pH 7.6
|-
! scope="row" style="background:#efefef;" |150mM NaCl
|200ul 0.5M EDTA
|-
! scope="row" style="background:#efefef;" |10mM EDTA
|200ul 0.5M EDTA
|-
! scope="row" style="background:#efefef;" |1% TritonX-100
|1 ml 10% TritonX-100
|-
! scope="row" style="background:#efefef;" |Protease inhibitors
|1 Roche complete mini tablet (4°)
|}


DAY ONE


:- Pre-prime the males and females with 10 units (0.1ml 100U/ml) of human chorionic gonadotropin (hCG) 20 hours before priming.


Lysis and SDS PAGE
DAY TWO


1. Turn on Lieb lab water bath biorupter about 30min before lysis.
:- Prime the males and females with 200 units (0.2ml 1000U/ml) of hCG.
:- Place each of the primed females into separate tanks containing 1x MMR to a depth of approximately 2 inches. Cover the tanks to avoid disturbing the frogs while they lay their eggs. Females should start to lay eggs within three hours of being primed.
:- Place each of the primed males into separate tanks of water.
:- Always ensure that the water temperature does not drop below 23˚C.
:- Collect eggs from the tanks using a plastic transfer pipette coated with L-15 Leibovitz medium containing 10% calf serum (this prevents the eggs from sticking).
:- Euthanize the male and dissect the testes into L-15 + 10% calf serum on ice. Avoid tearing the testes during dissection.
:- Transfer the required number of eggs into a Petri dish, gently spread them out to form a monolayer and remove any excess buffer.
:- Transfer testes into the Petri dish containing the eggs, together with a drop of L-15 + calf serum. Macerate the testes thoroughly using forceps, then gently mix the sperm and eggs. Grasp the macerated testis tissue with the forceps and gently ‘paint’ over the surface of the eggs a couple of times. Allow the eggs to sit for five minutes before flooding the dish with sterilized frog-system water (at room temperature).
:- Fertilization should be apparent (from pigment contraction) after about 10 minutes. The majority of the fertilized eggs should subsequently rotate within their vitelline membranes so that the animal pole is uppermost.
:- Remove the jelly coat from the eggs using 2% cysteine hydrochloride (in water, pH 8.0). Remove the water from the dish, replace with cysteine-HCl, then gently agitate the fertilized eggs with a stream of cysteine-HCl using a transfer pipette. Once the jelly coat is removed, the eggs should pack together closely. After de-jellying, remove the cysteine-HCl immediately and thoroughly rinse four times with sterilized frog-system water.
:- Replace water with buffer as necessary.
:- Remove any unfertilized eggs and dead embryos, as these have a severely adverse effect on the development of the healthy embryos.


2. Lyse embryos on ice in 100ul of cold lysis buffer by pipetting up and down.
10X MMR:


3. Sonicate samples for 10min on High setting: 30sec on/ 30sec off.
:0.1M NaCl
:2mM KCl
:1mM MgSO4
:2mM CaCl2
:5mM HEPES


4. Spin samples at 4° at 14,000 rpm for 5min.


5. Move supernatant to new tube.
The pH of the stock solution should be adjusted to 7.4 with NaOH.


6. Normalize loading volumes by performing a Coomassie protein assay for all samples.
Note. It’s important to be accurate in making this stock solution, and the final buffer dilution, because there are anecdotal reports of poor egg quality and low fertilization frequencies associated with MMR that is too highly concentrated.


7. For maximum loading volume combine 17.75ul protein (17.75ul of sample with lowest concentration, then normalize other samples to this using water to make up the difference) with 6.25ul 4X Loading Dye and 1ul BME. 


8. Boil samples for 6-10 minutes.
'''Protocol for X.tropicalis natural mating'''


9. Spin samples at 14,000 rpm, RT for 3 min.
Natural mating is a more efficient method of obtaining X.tropicalis embryos than IVF, generating significantly higher numbers (up to 2000), and is therefore better suited to genetic screening applications.


10. Load 25ul of sample onto gel for western analysis.
DAY ONE


:- Pre-prime the males and females with 10 units (0.1ml 100U/ml) of human chorionic gonadotropin (hCG) 20 hours before priming.


FOR TISSUE CULTURE:
DAY TWO


:RIPA Buffer (100 mL – can store at 4°)
:- Prime the males and females with 200 units (0.2ml 1000U/ml) of hCG.
:- Set up single-pair matings in tanks containing frog system water to a depth of approximately 2 inches. Always ensure that the water temperature does not drop below 23˚C.
:- Cover the tanks to avoid disturbing the frogs.
:- Monitor for amplexus and allow frogs to mate for several hours.
:- When the frogs stop mating, or when sufficient eggs have been layed, remove the frogs to a second tank.
:- Check samples of eggs/embryos from each tank for fertilization.
:- OPTIONAL If early developmental stages are required, embryos can be collected and sorted for continued culturing by rinsing them from the tank with 2% cysteine-HCl, transferring them to Petri dishes to finish de-jellying, then rinsing thoroughly with sterilised frog water before sorting healthy embryos from any unfertilized eggs or dead embryos.


:Add 790mg Tris base to 75mL diH20.  (50mM Tris-HCl, pH 7.4)


:Add 900mg NaCl and stir until all solids are dissolved. (150mM NaCl)
Raising X.tropicalis


:Using HCl, adjust pH to 7.4
:- Embryos from natural mating can be allowed to develop in the tank until hatching, when they will begin to attach themselves to the sides of the tank. At this point they should be transferred to a clean tank containing frog system water. Embryos should be cultured at between 25˚ and 28˚C. Lower temperatures slow their development and lead to increased death.
 
:- Add fresh water to the tanks daily and remove any dead embryos.
:Add 10mL 10% NP-40 (1% NP-40)
:- Air-stones should be included in the tanks to circulate and oxygenate the water.
 
:- Tadpoles reach feeding stage at 3 to 4 dpf, indicated by the appearance of small air bubbles at the surface of the water. Feed twice daily with a very fine suspension of powdered food (Nasco frog brittle or Sera micron).
:Add 2.5mL 10% Na-deoxycholate and stir until solution is clear (0.25% Na-deoxych.)
:- Tanks should be cleaned daily by pouring tadpoles into a separate container, cleaning the tank, then transferring the tadpoles back into it.
 
:- Fresh water should be added daily, splitting the tanks as necessary.
:Add 1mL 100mL EDTA. (1mM EDTA)
:- Tadpoles can be transferred to glass aquariums at around 7 to 10 dpf. The aquariums should contain an air-stone to oxygenate the water and a heater to maintain the water temperature at 26-27˚C. 20% of the water in the aquarium should be changed daily.
 
:Bring to 100mL with diH20.
 
 
At time of use, add protease inhibitors
 
Lysis and SDS PAGE
 
1. Aspirate media from cells. Wash cells with ice cold PBS 1X.
 
2. Add 200uL RIPA/well of 6-well dish (just enough RIPA to barely cover cells)
 
3. Rock plate at 4° for 5min.
 
4. Rinse cells off plate using RIPA buffer. Alternately, scrape cells from well in PBS, then spin at 1500 rpm for 10min at 4°, aspirate PBS from pellet, and resuspend pellet in desired amount of RIPA.
 
5. Transfer lysate to eppendorf. Sonicate samples for 5min (30 sec on/30 sec off) on High in Lieb lab biorupter.
 
6. Spin samples at 14,000 rpm for 5min at 4°.
 
7. Move supernatant to new tube.
 
8. Normalize loading volumes by performing a Coomassie protein assay for all samples.
 
9. Combine desired amount of protein (minimum of 10ug) up to 17.75ul (normalize all samples so that each samples contains the same amount of total protein) with 6.25ul 4X Loading Dye and 1ul BME.
 
10. Boil samples 6-10 minutes.
 
11. Spin samples at 14,000rpm for 3min at RT.
 
12. Load 25ul onto gel.
 
 
Transfer
 
1.  Build “gel sandwich” in transfer buffer using two pieces of Whatman paper, a nitrocellulose membrane, and two sponges.  I find it easiest to build using this orientation:  Gray side-sponge-Whatman paper-gel-nitrocellulose-Whatman paper-sponge-clear side.  Gray side faces black electrode! Don’t forget to orient the gel on the membrane so that the lanes do not transfer backwards.
 
 
Transfer Buffer
 
:15g glycine
 
:3.5g Tris base
 
:Bring to 800ml with diH20
 
:Add 200ml MeOH
 
 
2.  Transfer gel ON at 30V or for 1 hr at 400 mA at 4°.
 
3.  Transfer membrane to TBST for storage or to blocking solution.
 
 
Immunoblotting
 
1. Block membrane in 5% dry milk in TBST for at least 1 hour at RT or ON at 4°
 
2. Wash membrane 3X in TBST, 5-15min/wash
 
3. Apply primary antibody diluted in Blocking Solution ON at 4° or for 2hrs at RT.
 
4. Wash membrane 3X in TBST, 5-15min/wash
 
5. Apply secondary antibody diluted in 5% milk in TBST for 1h at RT.
 
6. Wash membrane 4X in TBST, 5-15min/wash.
 
7. Develop with ECL.

Latest revision as of 13:12, 22 November 2011

Protocol submitted by VGP from Conlon lab protocols [1]


link to protocol document [2]


Protocol for anaesthesia of adult X.tropicalis

Anaesthesia can be used to make the frogs easier to handle during sub-cutaneous injection of hCG, reducing the likelihood of injury to the animals.

- Place frogs into tanks containing frog system water to a depth of approximately 2 inches.
- Add ethyl 3-aminobenzoate methanesulfonate salt (Tricaine/MS222; Sigma) to a final concentration of 0.025% (w/v). Tricaine can be stored at 4˚C as a 2.5% stock solution in water.
- Allow approximately 30 minutes for the anaesthetic to take effect. Do not leave the frogs in the anaesthetic for longer than is necessary for anaesthesia.
- Once the procedure is complete, allow the frogs to recover from anaesthesia in shallow water (i.e. not more than 2 inches deep).


Protocol for in vitro fertilization of Xenopus tropicalis

This method should routinely result in >70% fertilization per batch of eggs. However, not all males are equally fertile, so two males should be primed per experiment to allow for a backup male should the first be infertile. Four females should allow selection of the best quality eggs and provide enough for several rounds of injections etc.

DAY ONE

- Pre-prime the males and females with 10 units (0.1ml 100U/ml) of human chorionic gonadotropin (hCG) 20 hours before priming.

DAY TWO

- Prime the males and females with 200 units (0.2ml 1000U/ml) of hCG.
- Place each of the primed females into separate tanks containing 1x MMR to a depth of approximately 2 inches. Cover the tanks to avoid disturbing the frogs while they lay their eggs. Females should start to lay eggs within three hours of being primed.
- Place each of the primed males into separate tanks of water.
- Always ensure that the water temperature does not drop below 23˚C.
- Collect eggs from the tanks using a plastic transfer pipette coated with L-15 Leibovitz medium containing 10% calf serum (this prevents the eggs from sticking).
- Euthanize the male and dissect the testes into L-15 + 10% calf serum on ice. Avoid tearing the testes during dissection.
- Transfer the required number of eggs into a Petri dish, gently spread them out to form a monolayer and remove any excess buffer.
- Transfer testes into the Petri dish containing the eggs, together with a drop of L-15 + calf serum. Macerate the testes thoroughly using forceps, then gently mix the sperm and eggs. Grasp the macerated testis tissue with the forceps and gently ‘paint’ over the surface of the eggs a couple of times. Allow the eggs to sit for five minutes before flooding the dish with sterilized frog-system water (at room temperature).
- Fertilization should be apparent (from pigment contraction) after about 10 minutes. The majority of the fertilized eggs should subsequently rotate within their vitelline membranes so that the animal pole is uppermost.
- Remove the jelly coat from the eggs using 2% cysteine hydrochloride (in water, pH 8.0). Remove the water from the dish, replace with cysteine-HCl, then gently agitate the fertilized eggs with a stream of cysteine-HCl using a transfer pipette. Once the jelly coat is removed, the eggs should pack together closely. After de-jellying, remove the cysteine-HCl immediately and thoroughly rinse four times with sterilized frog-system water.
- Replace water with buffer as necessary.
- Remove any unfertilized eggs and dead embryos, as these have a severely adverse effect on the development of the healthy embryos.

10X MMR:

0.1M NaCl
2mM KCl
1mM MgSO4
2mM CaCl2
5mM HEPES


The pH of the stock solution should be adjusted to 7.4 with NaOH.

Note. It’s important to be accurate in making this stock solution, and the final buffer dilution, because there are anecdotal reports of poor egg quality and low fertilization frequencies associated with MMR that is too highly concentrated.


Protocol for X.tropicalis natural mating

Natural mating is a more efficient method of obtaining X.tropicalis embryos than IVF, generating significantly higher numbers (up to 2000), and is therefore better suited to genetic screening applications.

DAY ONE

- Pre-prime the males and females with 10 units (0.1ml 100U/ml) of human chorionic gonadotropin (hCG) 20 hours before priming.

DAY TWO

- Prime the males and females with 200 units (0.2ml 1000U/ml) of hCG.
- Set up single-pair matings in tanks containing frog system water to a depth of approximately 2 inches. Always ensure that the water temperature does not drop below 23˚C.
- Cover the tanks to avoid disturbing the frogs.
- Monitor for amplexus and allow frogs to mate for several hours.
- When the frogs stop mating, or when sufficient eggs have been layed, remove the frogs to a second tank.
- Check samples of eggs/embryos from each tank for fertilization.
- OPTIONAL If early developmental stages are required, embryos can be collected and sorted for continued culturing by rinsing them from the tank with 2% cysteine-HCl, transferring them to Petri dishes to finish de-jellying, then rinsing thoroughly with sterilised frog water before sorting healthy embryos from any unfertilized eggs or dead embryos.


Raising X.tropicalis

- Embryos from natural mating can be allowed to develop in the tank until hatching, when they will begin to attach themselves to the sides of the tank. At this point they should be transferred to a clean tank containing frog system water. Embryos should be cultured at between 25˚ and 28˚C. Lower temperatures slow their development and lead to increased death.
- Add fresh water to the tanks daily and remove any dead embryos.
- Air-stones should be included in the tanks to circulate and oxygenate the water.
- Tadpoles reach feeding stage at 3 to 4 dpf, indicated by the appearance of small air bubbles at the surface of the water. Feed twice daily with a very fine suspension of powdered food (Nasco frog brittle or Sera micron).
- Tanks should be cleaned daily by pouring tadpoles into a separate container, cleaning the tank, then transferring the tadpoles back into it.
- Fresh water should be added daily, splitting the tanks as necessary.
- Tadpoles can be transferred to glass aquariums at around 7 to 10 dpf. The aquariums should contain an air-stone to oxygenate the water and a heater to maintain the water temperature at 26-27˚C. 20% of the water in the aquarium should be changed daily.
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