-
fix embryos with a fixative compatible with the antibodies to be used
Dent's and MEMPFA are all good choices - fix for 2 h at rt followed
by 30 min. in Dents
- Wash briefly
in PBS and soak in either 15% cold water fish gelatin/15% sucrose in
PBS overnight
or in 7.5% porcine gelatin
(300 Bloom) 15% sucrose in PBS for 6 h at 37°C.
- Embed in
15% sucrose/7.5% gelatin, chill to 4°C (you can hold the samples
here for a few days).
- Remove sucrose/gelatin
solution and add Tissue-Tek OCT (Miles Scientific, Naperville, IL 60566),
compound,
allow to equilibrate for at least 30 min (it can stay in there longer).
- Prepare
a block using Tissue-Tek.
By controlling temperatures, it is then possible
to position your sample on the block in the desired orientation.
Typically this is done using a stereomicroscope and forceps.
Once in position, re-freeze
the block, either in a -80°C freezer on using a dry ice/ethanol
bath.
- Place the
block into the cyrostat and allow it to equilibrate to the cutting temperature
(i.e. -17 °C)
- Prepare
12-14 µm thick sections using a cryostat at -17°°C and
collected onto pre-coated
or frosted glass slides and
store at -80°C until you are ready to stain them.
(Colorfrost®/Plus - Fisher Scientific
Co). Warm slides and allow them to dry at room temperature
- extract
for 2 min. in 100% acetone.
- rehydrate
in PBS
- block with
TNB blocking buffer (from tyramide kit) for 30 min at r.t. or just incubate
in PBS + 0.5% Tween20
incubate in primary antibody (2 h at 16°C or overnight)
- rinse PBS
containing 0.5% Tween 20
- incubate
with secondary antibody (2h at 30°C)
*** typically we are now
using ALEXA-conjugated secondary antibodies from Molecular
Probes.
- wash in
Tween PBS
- mount in
airvol + propyl gallate
- examine
by standard, deconvolution or confocal microscopy
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Table of Methods