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Whole Mount RNA In-Situ Hybridization  

In-situ hybridization is when one uses a probe to detect either DNA or RNA sequence inside cells.

1X PBS (Phosphate buffered saline)
1X PBT (50 ml 1X PBS + 250 ul 20% Tween-20)
100% Methanol
Proteinase K (in ice bucket)
Formamide (in ice bucket)
5 mg/ml heparin (in ice bucket)
50 mg/ml tRNA (in ice bucket)
I M citric acid (in ice bucket)
RNase-free water
20% Tween-20
100 mg/ml BSA (in ice bucket)
Sheep serum (in ice bucket)
4% PFA***** (in ice bucket: 2 g paraformaldehyde dissolved in 50 ml 1X PBS)

Chemical safety:
The other chemicals used in the protocol are not notably dangerous.  Like with most chemicals, if the chemicals or solutions used in this lab come in contact with your eyes or skin, you should flush the affected area with copious amounts of water, and tell the instructors about the spill.  If a chemical comes in contact with your eyes, or if you have any ill effects from contacting a chemical, you should call a physician. 

Gloves must be worn throughout the first day of the laboratory.  This is both to protect you from any chemicals and to protect the sample.  RNAses, which will degrade the single stranded RNA probe, are present on your hands.  If you touch the tube containing the probe, for example, the whole experiment could fail.

Gastrula stage embryos are very fragile; please pipet very carefully.  You may have no tissues left at the end if you don't follow this advice.

Never let the embryos get completely dry-they will get very sticky and start to break.  Always leave a little bit of the previous solution on.

In situ hybridization, day 1

Set up

You should pipet each set of embryos you are planning to use into 1.5 ml eppendorf tubes.  It is not wise to put more than ~30 embryos into one tube, or they might not all get exposed to the probe.  You might have to split some samples into more than one tube.

The needed solutions will be stationed at several points in the laboratory, or will be available in an ice bucket or freezer.


To change a solution (this is true for whole protocol): 
Remove most of the solution on the embryos using a pasteur pipet (but make sure to leave enough so the embryos don't get dry).  Slowly pipet about 1 ml of the next solution onto the embryos.  Don't just squirt the liquid in, or your embryos will be gone by the end.  Place the pipet up gently up against the side of the tube, and pipet the new solution out slowly.  If you are moving the embryos from one tube to another, make sure that none of the embryos are left in the pipet.

Rehydrate embryos:
Make the following solutions in 50 ml conical tube.  Make 40 ml of each.  Measurements do not have to be very precise-you can use the gradations on the side of the conical tube if you want to.

75% methanol/25% PBS (30 ml methanol + 10 ml PBS)
50% methanol/50% PBS
25% methanol/75% PBS

Incubate each sample 5 minutes at room temperature with 75% MetOH/25% PBS, then 50% MetOH/50% PBS, then  25% MetOH/75% PBS

Equilibrate into PBT:
Incubate 5 minutes at RT with PBT
repeat three times (for a total of four washes in PBT)

If you are using embryos younger than 24 hours post fertilization, stop here and go to step 4

1. Permeabalize embryos:

Older embryos need to be treated with Proteinase K (PK), an enzyme that chews up protein, to make holes in the cells of the embryo so the probe can get in. Come get the instructor before you start the PK incubation, so the instructor can be ready to add the PFA. If the PFA does not get added promptly, your embryos might dissolve.

Make a 5 ug/ml solution of PK in PBT:
Add the correct volume of a concentrated PK solution into 5 ml of PBT in a 15 ml conical tube.

PK times
        1 cell stage to -24 hours post-fertilization:  0 minutes
        24 hours-48 hours post-fertilization: 10 minutes
        older than 48 hours post-fertilization:  20 or more minutes

Add the PK solution to the appropriate sample and incubate for exactly the right amount of time

After PK treatment, immediately take off the PK and the insructor will add 4% PFA. *****PFA is dangerous.

Incubate in the 4% PFA for 20 minutes.

2. The instructor will remove the PFA**** to a waste container.  The instructor will do one wash with 1X PBT and remove this to waste container.

3. Wash embryos that have been PK treated 4 times for 5 minutes with PBT.

4. Make 50 ml Hybridization mix in a 50 ml conical tube. One batch of this should be enough for a group of four students.

Hybridization mix (50 ml total):
Formamide                                                             25 ml           
20X SSC                                                             12.5 ml           
heparin (5 mg/ml)                                                  0.5 ml                       
tRNA (50mg/ml)                                                  0.5 ml                       
citric acid (1M)                                                   0.46 ml           
RNase-free water                                           10.7 ml           
20% Tween-20                                           0.25 ml     

5. Equilibrate ALL embryos in Hybridization mix (this is also called the prehyb step).  Remove last PBT wash and put on about 500 ul of Hyb mix.  Place in tube rack in 70 degree C water bath for 45 minutes.

6. Hybridize embryos with probe.  Remove prehybridization solution.  Add 200 ul of fresh Hyb mix and 1 ul of the probe to each sample.  Probes should be labeled with Digoxegenin (DIG) or Fluorescein (FITC).

7. Hybridize overnight at 65-70˚C.  The instructor will take them out the next day and put them in the refrigerator. 

In situ hybridization, day 2

1a. Make 20 mls of the following solutions in a 50 ml conical tube.

2X SSC (2 ml 20X SSC + 18 ml water)
0.2X SSC (2 ml 2X SSC + 18 ml water)
50% Hyb Mix/50% 2X SSC (10 ml Hyb Mix + 10 ml 2XSSC

Put these three solutions in the 70 degree water bath for about five minutes to warm up.

1b. Make 20 mls of the following solution

50% 0.2X SSC/50% PBT (10 ml 0.2XSSC + 10 ml PBT)

2. Equilibrate the samples into SSC buffer:
15 min 50% HM-50% 2X SSC at 70˚C
15 min 2X SSC at 70˚C

3. Wash away non-specifically-bound probe:

Incubate 2 times for 30 min in 0.2X SSC at 70˚C

4. Equilibrate into PBT:
  5 min 50% 0.2X SSC/50% PBT at RT
  5 min PBT at RT

5. Make 10-20 ml of PI buffer in 15 or 50 ml conical tube
PI buffer:
10 or 20 ml PBT
200 or 400 ul  BSA (100 mg/ml)
200 or 400 ul sheep serum

6. Block non-specific sites on embryo

Incubate embryos in PI buffer (recipe above) at RT for at least 1h. The proteins in the PI buffer will bind to any "sticky" places on the embryo.  This is called blocking.  It prevents the antibody from binding to places that it shouldn't.

7. Dilute antibody 1:5000 in PI buffer.  Make 1 ml of solution for each sample. (For example, for 10 samples add 2 ul antibody in 10 ml PI buffer in 10 ml conical tube). Add 1 ml of diluted antibody to each sample.  The instructor will put them to incubate at 4˚C overnight on a shaker.

In situ hybridization, day 3

1. Do several ~ 1 ml washes with PBT:
-one quick wash in PBT
-5 X 15 min washes in PBT at RT on rocker

2. While the washes are going, make 50 ml Alkaline Phosphatase (AP) buffer in a 50 ml conical tube

AP buffer                                                            for  50ml:
100 mM Tris pH 9.5                                             5 ml of 1M
50 mM MgCl2                                                   2.5 ml of 1M
100 mM NaCl                                                       1 ml of 5M
0.1% Tween-20 (Bring to 50 ml with water)         0.25 ml of 20% Tween-20

Note: it is best to use AP buffer that has been made within the last week or so.  It should be stored at 4˚C in between uses.

3. Equilibrate embryos into AP buffer
            -one quick wash in AP buffer-it is important to do this quickly-or a precipitate will
            -three 5 minute washes in AP buffer

4. During the washes, make 5 ml developing solution in a 15 ml conical tube. 
Note: This solution is light sensitive; wrap tube in aluminum foil.

Developing solution                       
5 ml AP buffer
11.25 ul NBT (100mg/ml)
17.5 ul BCIP (50mg/ml)

5. Remove AP buffer from each sample, and add a little bit less than 1 ml developing solution.  Put each sample of embryos in one well of a glass histology dish (make sure to label well!).  Place in the dark (under a box or in a drawer) to develop.  You can also place developing embryos at 37 ˚C to make the color development go more quickly. Be careful, as the background staining will also come up more slowly.

6.  Monitor color development under dissecting microscope with an overhead light, and white base under your dish.

The color will develop at very different rates depending on which probe was used. 
A good rule of thumb is to check the embryos at 5 minutes, and then 10 minutes.  If nothing has come up at 10 minutes, you are safe to wait another 20 minutes to check again, and so on.  Some probes are very slow, and may not even be done by the end of class.

If you have no staining at the end of class, move the embryos to an eppendorf tube to continue developing.  You have three choices:
-If they have no color at all, you can add fresh developing solution, and incubate
            them overnight in the refrigerator.
-If they are beginning to have a little color, you can add half strength developing solution (1 part developing solution, 1 part AP buffer) and let them incubate overnight in the refrigerator.
-If they are almost done, you can rinse them with AP buffer, and incubate them overnight in AP buffer.  They will continue to develop at a very slow rate.

7. Stop reaction by several quick washes in PBT.  Store in PBT in the dark at 4˚C.