HELP-zebrafish antibody staining after day3 - (Oct/28/2012 )

I am doing antibody staining with zebrafish embryos after day2 or day3 . But I find that it is very difficult to get a good reslt .

if you know an good way to do it, could you let me know?

thanks!

Some details would help - antibodies, times, blocking, washing, fixing...

Otherwise your post reads: "I'm having a problem, help!"

bob1 on Sun Oct 28 19:15:06 2012 said:

Thank you for your reply,Do you have a protocol ?

Not specifically for zebrafish, and very antibody dependent, but yes, I do have IF protocols....

However, you said that you had a problem, if you can give your protocol, then we can try to help you troubleshoot, or may know a (quick) fix for a certain problem.

bob1 on Mon Oct 29 04:18:07 2012 said:

this is our protocol


Antibody Staining Protocol for Zebrafish

Reagents:
PT:
PBS + 0.3% Triton X-100

PBTN:
PT + 4% BSA + 0.02% NaN3

PEM:
0.1 M PIPES, 1.0 mM MgSO4, 2 mM EGTA, pH to 7 with NaOH

Fixative:
PEM + 2-4% formaldehyde


1. Wash 15-40 embryos (or larvae) 2-4 times in 1 ml PT.
2. Wash 2 times in 1 ml PBS.
3. Add 1ml of fixative (PEM + 2-4% formaldehyde or use 2-4% PFA in PBS). Note: Make fresh fixative each time by adding formaldehyde (37%) to stock PEM solution. Fix at 4 °C for 2 hours in 4% formaldehyde or 4 hours to overnight in 2% formaldehyde on shaker (I prefer 2% formaldehyde in PEM overnight).
4. Wash 3-5 times in PBS. Remove yolk if necessary (recommended for gut and endodermal organ stainings). Block with 1 ml of PBTN for an hour at 4 °C.
5. Add primary antibody diluted in 100 ul PBTN. Incubate overnight to 48 hours at 4 °C on shaker (I prefer overnight). Note: Lay the tube on the shaker, but with a slight slope to make sure that all the solution is at the bottom of the tube.
6. Recover antibodies and save (can be reused for 3 times). Wash 5 times in 1 ml of PT with 30-45 minutes each.
7. Add appropriate secondary Alexa fluorescent-conjugated antibodies diluted (normally 1:500-1:2000) in 200 ul PBTN. Incubate for 2 hours at room temperature or 4 hours to overnight at 4 °C on shaker (I prefer overnight). Note: Once the fluorescent secondary antibodies are applied, the tube shoule be protected against light during incubation.
8. Remove antibodies. Wash 5 times in 1 ml of PT with 30-45 minutes each.
9. Mount the embryo on slide in Vectashield or Mowiel, or immerse into 80% glycerol in PT for microscopy. If the embryos needs to be stored for several days to two weeks before microscopy, do a secondary fix of 4% formaldehyde in PBS for 1 hour at room temperature. Embryos can be stored in PT at 4 °C for up to two weeks.

That looks like a quite standard protocol, apart from the wash durations, which may be too long and too stringent - you can permeablize in the PT buffer and then do the washes in PBS-T (PBS with 0.1% tween 20).

If you are having significant background you may need to use a different fixative with a the ability to decolourize the cells - this is often glutaraldehyde (instead of formaldehyde) and DMSO based, but I can't remember the specific details or even the name of the solution.

You might be well advised to have a look on IHCworld.com, where there are heaps of protocols and basic explanations of different solutions and techniques.

bob1 on Mon Oct 29 07:42:12 2012 said:

Thank you very much