Protocol - Sea urchin whole mount immunostaining
localization of antigens in specific territory during sea urchin development
Category:
In situ hybridization and Immunochemistry
Last revision: Dec 16, 2014
Contact
Name: Marta Di Carlo
Address: IBIM-CNR, via Ugo La Malfa, 153, Palermo
Phone: 0916809538
Email: di-carlo@ibim.cnr.it
Figure legend:
P. lividus pluteus larva incubate with anti-U295, an antiboby reconizing neuronal cells
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
|
Embryo fixation
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|||
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Wash the embryos in seawater 3 times
|
R.T.
|
10 minutes
|
|
|
Fix embryos in 1% glutaraldehyde, 4% formaldehyde in seawater
|
R.T.
|
30 minutes
|
continuous gentle mixing
|
|
Wash the embryos in seawater 2 times
|
R.T.
|
10 minutes
|
|
|
Resuspend embryos in ethanolammina 1M pH8
|
R.T.
|
3 minutes
|
|
|
Wash the embryos in seawater 2 times
|
R.T.
|
10 minutes
|
|
|
Remove supernatant and resuspend embryos in a small volume of seawater, add 100% methanol
|
R.T.
|
3 minutes
|
store at -20°C
|
|
Rehydrate in 50% methanol in PBST 1X
|
R.T.
|
3 minutes
|
|
|
Wash the embryos in PBST 1X 2 times
|
R.T.
|
10 minutes
|
|
|
Incubate embryos in 3% BSA serum (goat) in PBST 1X
|
R.T.
|
30-60 minutes
|
with gentle mixing
|
|
Dilute the primary antibodies at the specific concentration in 3% BSA in PBST 1X
|
R.T.
|
3 minutes
|
|
|
Centrifuge the embryos at 600 rpm
|
R.T.
|
2 minutes
|
discard supernatant
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|
add primary antibodies at the established dilutions
|
4°C
|
over-night
|
|
|
Wash the embryos in PBST 1X 3 times
|
R.T.
|
10 minutes
|
|
|
Add secondary antibody, anti-rabbit Alexa 594 or anti-mouse devices Alexa 488, diluted in 1% BSA in PBST 1X
|
R.T.
|
1 hour
|
incubation in the dark
|
|
Wash the embryos in PBST 1X 3 times
|
R.T.
|
10 minutes
|
|
|
Transfer the embryos in 35% glycerol in PBST 1X
|
R.T.
|
2 minutes
|
|
|
Mount embryos on a slide and observe at fluorescent microscope
|
R.T.
|
10 minutes
|
Quality validation:
Yes
Validation info
The protocol has been used to obtain data published in a peer-reviewed journal.
Solutions
-
PBST 1X
Reagents F-concentration Quantity I-concentration KCl2,7 mM10 ml/L27 mMNaCl137 mM10 ml/L1.37 mMNa2Po4, pH 7,48 mM10 ml/L80 mMKH2PO41.9 mM10 ml/L19 mMTriton X1000,2%2 ml/L100%
