with specific antibodies that are detected by enzymatic reactions
(alkaline phosphatase and peroxidase). So far, this technique has
been mostly developed for light microscopy methods that do not
inform on the surrounding ultrastructure, such as the presence of
cytoskeletal elements, organelles, or membrane-less assemblies. To
allow for the ultrastructural analysis of mRNA localization at the
EM level, we have developed a post embedding method combining
ISH with EM, that we called ISH-IEM [5]. Of note, this is not the
first EM method to be developed (seereferences in [5]), but the one
presented in this article allows the simultaneous visualization of
mRNAs and proteins on the same frozen section [6, 7].
Although we use frozen sections to preserve protein antigenic-
ity, the cellular morphology is somewhat compromised and one
important aspect of this ISH-IEM method is to postfix the frozen
sections before their hybridization.
The fixed frozen sections are then hybridized (ISH) with DIG
or biotin labeled antisense RNA probes overnight in a humid
chamber. This allows an efficient hybridization (transfer and
annealing of the antisense probe to the target mRNA). At least
forgurkenmRNA inDrosophilaegg chambers, the optimal tem-
perature is 55C, and addition of dextran sulfate to the hybridiza-
tion buffer allows for a more efficient detection [5].
The tagged probes are then detected by immunoelectron
microscopy (IEM) using anti-DIG or anti-biotin antibodies.
Those are visualized by protein A conjugated to colloidal gold
particles (PAG) according to the protocol developed by [4].
As protein antigenicity is retained in frozen sections, they can
be detected using specific primary antibodies together with
mRNAs, as performed for classical double immunolabeling [4].
We used this protocol ongurkenmRNA andbicoidmRNA
both localized at the (dorso)-anterior corner of the stage 9 Dro-
sophila oocyte and successfully showed that there were localized to
P-bodies (Fig.1)[5–7]. Note that the procedure that is described
below can easily be adapted to fluorescence detection on thick
sections [5].2 Materials
Many reagents are toxic and should be handled with care in the
fume hood and using gloves.2.1 Fixatives (See
Notes 1 and 2)
- 4% paraformaldehyde (PFA): prills 95 w/v % to be diluted in
0.1 M phosphate buffer (not PBS) at pH 7.4 to make a 16%
stock that can be frozen. Prepare 4% solution fresh from frozen
16% stock.
178 Catherine Rabouille