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- This protocol is the one we give to starting researchers in our lab so it is detail heavy. It is current for our lab as of 1-Nov-09 The protocol is based off of the Thisse in situ protocol found here: http://zfin.org/ZFIN/Methods/ThisseProtocol.html
- We find that we don't don’t actually have to be that finicky about RNAse-free conditions. We are very careful when we are making up the probe (i.e., in vitro transcription), but we find that in situs turn out fine when we simply use standard cleanliness during the actual protocol. However, most people have dedicated stocks of solutions that are just used for in situs and dedicated plastic transfer pipets (which can be re-used) for the various steps of the in situs. If you were concerned about being RNAse-free, it is mainly important for day 1, before the embryos get into HYB solution.
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- In all steps, take care not to mix heavily or allow air interface contacts with the embryos. When the protocol calls for "washing" “washing” embryos, add 1000 ul of solution to side of the eppendorf tube. This results in gentle mixing of the embryos with the wash solution. Don't Don’t squirt solutions directly onto the embryos. If needed the tubes can be given a mild extra mix with a gentle inversion to make sure that the solution is equally distributed among the embryos and that they are not stuck to each other. Since you are trying to keep them intact over 3 days of washing, its important to be gentle.
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Purpose of day 1: Prepare the embryo for hybridization with DIG probe by re-hydrating and poking holes (with proteinase K) to let probe in. Hybridize embryos with DIG probe.
1. Wash at RT:
5' 5’ in 75% MeOH: 25% PBT
5' 5’ in 50% MeOH: 50% PBT
5' 5’ in 25% MeOH: 75% PBT
We do these exchange washes to gently re-hydrate them into PBT. The embryos are very brittle in MeOH. If you put the embryos directly into PBT, they are more likely to break up. Other labs do two 5' 5’ washes using 66%/33% and 33%/66% solutions. We make these exchange solutions up fresh right before use.
2. Wash 4x in 100% PBT for 5' 5’ at RT.
3. Incubate embryos in proteinase K at RT.
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Most protocols recommend: 5' 5’ somite stages; 10' 10’ for 24hrs +; 20' 20’ for 48 hrs +. But, each new proK batch has to re-optimized because the potency of each batch varies. Recently, we've we’ve used: 30 seconds somite stages; 2.5' 5’ for 48 hours; 5' 5’ for 3dpf.
Be careful not to over-proteinase! This will cause your embryos to fall apart. Make sure you have the 4% PFA thawed and ready to go before you start the proK step.
4. Re-fix embryos in 4% PFA for at least 20' 20’ at RT.
This step stops the reaction because PFA kills the ProK. Make sure you mix gently to ensure that all embryos are exposed to PFA; you can also lie the tubes on their side, evenly distributing the embryos in the solution. This PFA does not have to be fresh (it can be in the fridge for up to 2 weeks). Don't Don’t let this step go too long (the most people have tried in this lab is 1 hour).
5. Wash 5x in PBT for 5' 5’ at RT.
6. Prehybridize embryos in 500ul HYB buffer for at least 2 hours at 68°C. A longer prehyb is ok, and can even be better.
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When you remove the probe on Day 2, save the probe for future re-use. In our hands, we've we’ve found that people have been able to re-use probe for at least 5x. Keep track of how many times you've you’ve re-used probe so you know how long it takes for it to become too weak.
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Purpose of day 2: Wash off any excess DIG probe from embryos. Inactivate any endogenous alkaline phosphatase to reduce background (in older embryos, non-specific stain is seen in the ear, liver, and somites). Incubate embryos with antibody.
1. Wash at 68°C:
10' 10’ 75% HYB: 2x SSC10
' 10’ 50% HYB: 2x SSC
10' 10’ 25% HYB: 2x SSC10'
10’ 2x SSC
30' 30’ 0.2x SSC30'
30’ 0.2x SSC
Preheat all wash solutions to 68°C in the hybridization oven before starting washes (for our incubators, it takes 15 minutes for the incubator to warm to 68°C and another 45 minutes for the solutions to reach 68°C). We are slowly exchanging the embryos into SSC to be extra-gentle with them and prevent them from floating as you change from HYB to SSC. We make the exchange solutions up fresh right before use. Because of the salt in the SSC solutions, avoid keeping the solutions for too long in 68°C. The duration of these washes matter for stringency (see below), so don't don’t let them go over. Since the temperature of these washes also matter, take out the solutions from 68°C immediately before you are adding on the solution.
To adjust the stringency of these washes: For higher stringency (to reduce background for great probes), you can go up to 70°C, or go down to 0.05x SSC for the final wash. For lower stringency, you can go down to 65°C.
2. Wash at RT:
5' 5’ 75% 0.2x SSC: PBT5
' 5’ 50% 0.2x SSC: PBT
5' 5’ 25% 0.2x SSC: PBT5'
5’ PBT
We are slowly exchanging the embryos into PBT to be extra-gentle with them. Other labs do two 5' 5’ washes using 66%/33% and 33%/66% solutions.
3. Optional step for >48hpf embryos to remove endogenous AP.
Incubate 45' 45’ in 0.1M Glycine pH2.2 + 0.1% Tween at RT.
Wash 3x 5' 5’ in PBT at RT.
Glycine solution does not last long at RT. When we've we’ve made 1M stocks, they have developed precipitation after a month. They last longer at 0.1M.
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Start with a stock solution of PBT/BSA. This is PBT with BSA added to a final concentration of 2mg/ml. Make this solution fresh every time since BSA doesn't doesn’t keep well and stuff will grow in it. We typically make enough for d2 and d3 steps and store PBT/BSA at 4°C O/N. PBT/BSA formula: 0.02g BSA per 10ml of PBT. _Block formula:_50ul 100% NSS per 1ml PBT/BSA.
5. While embryos are blocking, make sure you have enough pre-absorbed anti-DIG Fab fragments for the antibody step (see pre-absorbing antibody protocol). This is standard but sometimes you use other antibodies such as anti-fluorescein-AP.
Other labs don't don’t bother pre-absorbing anti-DIG. They just make up the 1:2000 fresh.
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Formula: 10ul of 1:100 pre-absorbed stock per 200ul of block.
You don't don’t have to use block solution; you can also use PBT/BSA without NSS. Mix gently to evenly distribute the antibody solution; you can also lie the tubes on their side, evenly distributing the embryos in the solution.
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1. Wash for 1.5 hours in PBT/BSA, rocking, at RT. Do 1 quick changes plus 6x 15' 15’ washes.
This step is done on a rocking platform to help move the washes over the embryos.
With antibody washes, it is more the number of washes than the length of washes. You can leave the embryos in wash for longer than 15'15’, but then you should proceed to your next wash as if it was the normal time. For greater stringency, you can do more washes.
2. Wash 3x 5' 5’ in NTMT buffer at RT.
3. Stain with NBT/BCIP, in the dark, at RT.
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I prefer to stain in epitubes as you don't don’t have to worry about evaporation. Plus it is easy to keep track of large numbers of staining embryos when they are in separate tubes. If staining in tubes, add 1ml of staining solution. Lie the tubes on their side, evenly distributing the embryos in the solution. Place tubes in a drawer or in a box so they are protected from the light at all times.
If staining in dishes, you can see the embryos clearly, and you can use less staining solution, but you have to be very careful to keep track of which embryos are in which well. If you stain in a dish, make sure dish is really clean. Transfer embryos in lots of NTMT into a well. You can mark outside the well with a marker or a piece of tape to keep track of which embryos are in which wells. Remove excess NTMT and add 500ul of staining solution. Cover the dish so the embryos remain in the dark during the staining step! You can use a box or aluminum foil to cover the embryos. Make sure embryos don't don’t dry out and add more staining solution if necessary to prevent this.
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4. Stop the staining reaction by removing staining solution with 3x quick changes of NTMT at RT.
5. Wash in PBT for 5' 5’ at RT.
6. Fix embryos O/N in 4% PFA at 4°C. This PFA does no have to be fresh.
7. The next day: Wash 2x in PBT for 5' 5’ at RT. Score embryos. For long-term storage (at least several years), gradually equilibrate from PBT into 100% MeOH and store at --20°C. Some leaching of the stain may occur, but I have never found this to be a problem.
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