Zebrafish in situs

Starting notes:

-  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 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.

- Even though the protocol is always quite standard, it is a good idea to jot brief notes about how the experiment proceeded, especially noting variance from standard protocol and lengths of time used for incubations that can be variable (i.e. how long in PFA at 4 degrees before methanol treatment, how long in ProK, how long in pre-hyb, how long staining took). The most important thing is to keep track of which embryos were used with which probes. You can tape an in situ checklist in your notebook and check off the standard steps. Write onto the sheet any variances, and any notes.

- In all steps, take care not to mix heavily or allow air interface contacts with the embryos. When the protocol calls for “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 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.

- Reading up on a Northern protocol is a good point of reference to better understand the purpose of all the steps in the protocol. The in situ and the Northern protocols mirror each other because both use RNA anti-sense probes to target RNA.

Protocol:

Day 1 of the in situ:

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’ in 75% MeOH: 25% PBT

5’ in 50% MeOH: 50% PBT

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’ 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’ at RT.

3.      Incubate embryos in proteinase K at RT.

Working solution is 10ug/ml Cf made from 1mg/ml in PBT. Make sure you grab 1mg/ml stock, not 10mg/ml stock used for making DNA plates. Formula: 10ul of 1mg/ml stock per 1ml of PBT.

Most protocols recommend: 5’ somite stages; 10’ for 24hrs +; 20’ for 48 hrs +. But, each new proK batch has to re-optimized because the potency of each batch varies. Recently, we’ve used: 30 seconds somite stages; 2.5’ for 48 hours; 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’ 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 let this step go too long (the most people have tried in this lab is 1 hour).

5.      Wash 5x in PBT for 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.

Use different percentage HYB solutions to decrease background. For good probes that develop well, use 50% HYB. Some finicky probes will only come up in 60% HYB – but be prepared to see worse background developing in 60% HYB. 

7.      Replace preHYB with HYB buffer containing ~150ng probe in 200ul of HYB. Incubate O/N at 68°C.  

Formula: Typically, 5ul of stock probe from an in vitro transcription reaction in our lab is diluted in 200ul of HYB.  You can adjust the temperature down to 65°C or up to 70°C (see below). 

When you remove the probe on Day 2, save the probe for future re-use. In our hands, we’ve found that people have been able to re-use probe for at least 5x. Keep track of how many times you’ve re-used probe so you know how long it takes for it to become too weak. 

Day 2 of the in situ:

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’ 75% HYB: 2x SSC

10’ 50% HYB: 2x SSC

10’ 25% HYB: 2x SSC

10’ 2x SSC

30’ 0.2x SSC

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 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’ 75% 0.2x SSC: PBT

5’ 50% 0.2x SSC: PBT

5’ 25% 0.2x SSC: PBT

5’ PBT

We are slowly exchanging the embryos into PBT to be extra-gentle with them. Other labs do two 5’ washes using 66%/33% and 33%/66% solutions.

3.     Optional step for >48hpf embryos to remove endogenous AP.

Incubate 45’ in 0.1M Glycine pH2.2 + 0.1% Tween at RT.

Wash 3x 5’ in PBT at RT.

Glycine solution does not last long at RT. When we’ve made 1M stocks, they have developed precipitation after a month. They last longer at 0.1M.

4.      Block embryos with PBT/BSA + 5% normal sheep serum for 2 hours, rocking, at RT.

This step is done on a rocking platform to help move the block over the embryos.

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 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 bother pre-absorbing anti-DIG. They just make up the 1:2000 fresh.

6.      Incubate blocked embryos in 200ul pre-absorbed antibody*. Incubate O/N in the 4°C.

Formula: 10ul of 1:100 pre-absorbed stock per 200ul of block.

You 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.

*We typically use anti-DIG antibody at 1:2000. The Thisse protocol uses as little as 1:10,000. Anti-fluorescein antibody is diluted 1:1000.

Alternate protocol: You can incubate for 4 hours, rocking, at RT to do day 2+3.

Day 3 of the in situ:

Purpose of day 3: Wash off any excess antibody from embryos. Stain the embryos.

1.      Wash for 1.5 hours in PBT/BSA, rocking, at RT. Do 1 quick changes plus 6x 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’, 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’ in NTMT buffer at RT.

3.      Stain with NBT/BCIP, in the dark, at RT.

Formula: Use 2.5ul of 100mg/ml NBT and 3.5ul of 50 mg/ml BCIP per 1ml NTMT.

Make staining solution fresh every time!

You can stain in epi-tubes, or you can transfer to a staining dish.

I prefer to stain in epitubes as you 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 dry out and add more staining solution if necessary to prevent this.

Staining incubations are done at RT and must be done in the absence of light to keep background low. If a stain is taking a long time to come up, you can incubate at 37°C, but just be sure to check the stain more often.

Check embryos every 2 hours. Each tube will develop differently so it is important to look at embryos from each tube before you stop the reaction. If they need to go for longer, add fresh staining solution. You should let the staining reaction go a bit longer to make sure the stain is dark enough to take pictures; some of the background will be removed during the clearing step or by flat-mounting the embryos.

4.      Stop the staining reaction by removing staining solution with 3x quick changes of NTMT at RT.

5.      Wash in PBT for 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’ 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.

Reagents:

Day 1:

PBT = 1x PBS + 0.1% Tween-20. Store at RT.

100ml  10X PBS

900ml  Millipore water

1ml     100% Tween--20 (add slowly, very viscous)

 1L

Proteinase K:

Proteinase K. A 100X stock is 1 mg/ml in PBT. Make 1ml aliquots. Store at -20°C.

4% paraformaldehyde/PBS:

Old way with powder: Add 4g PFA in 100mls PBS. Dissolve at 68˚C. You must heat PFA to get it into solution. Let the solution come to RT.

New way with 20% solution from EMS: Add 100ml 20% solution to 400ml PBS.

Then: Check pH. It should be at 7.4. If not, adjust to correct pH with NaOH or HCl. Make 1ml, 5ml, and 10ml aliquots. Store at -20˚C.

Hybridization buffer:

Use dedicated bottles and solutions to ensure this is made RNAse free.

#                Stock                                   Stored at           Stock Source                                            Final

500ml        formamide                             -20˚C               Invitrogen 500g 15515026                              50%*

250ml        20x SSC                                 RT                                                                                   5x SSC

0.5g           torula (yeast) RNA                -20˚C               Sigma 100g R-6625                                      500ug/ml

0.05g         heparin                                    RT                  Sigma H-7005                                           50ug/ml

1ml            Tween                                     RT                                                                                    0.1%

4.6ml         1M citric acid pH6.0-6.5            RT                                                                                    9mM

  1L

* Formamide percentage can go up to 65% for higher stringency, but we typically only make 50% and 60% HYB. When thawed, the formamide is always <500ml, so you will need to scale down the recipe accordingly.

Make 50ml aliquots. Store at -20°C.

Day 2:

100% NSS:

Sheep serum comes from Jackson Immunologicals. Using a syringe, add 10ml of PBS to bottle of Normal Sheep Serum (make sure bottle says that it is meant to make 10ml of solution). Re-suspend. This makes a 100% stock. Make 1ml aliquots. Store at -20°C.

DIG Antibody:

Anti-DIG Fab Frag-AP conjugated 150u from Roche catalog# 11093274910. Store at 4°C.

Day 3:

NTMT (Staining Buffer):

Make fresh every day.

             #                     Stock                                      Final

            83ml                Millipore water

            10ml                1M Tris-CL pH9.5                       0.1M

            2ml                  5M NaCl                                    0.1M

            5ml                  1M MgCl2                                  0.05M

            100ul               Tween                                        0.1%

            100ml

            Some labs recommend including 1mM (final) levamisol to help inhibit endogenous Alkaline Phosphatase

NBT and BCIP:

NBT and BCIP are already prepared and purchased from Roche. This is light sensitive so use gloves and go in and out of them quickly.  

In the staining solution you want a final concentration of 225ug/ml of NBT and 175 ug/ml of BCIP based on the Thisse protocol. Jowett calls for more NBT (335 ug/ml) so the exact amount may not be critical.