as copper dissolves in the hybridization buffer. Sections might
fold or be lost from the grids during the procedure and this can
be fixed by preparing many grids for each labeling [5]- Make sure the humid chamber remains moist for the entire
procedure. This is critical. Grids should not dry out. If neces-
sary, add filter papers to the chamber and an excess of 50%
formamide [5] - Hybridization might lead to a loss of morphology because of
harsh extraction during formamide incubation and to a low
immune-reactivity. This can be solved by cutting thicker sec-
tions, and post-fixing them harder (with GA or acrolein), but
be aware that it might, for some mRNAs, lead to near or
complete loss of detection). The choice of fixatives depends
on the antigenicity of the protein you want to visualize with the
mRNA. - The melting temperature (Tm, at which 50% of the RNA mole-
cules are single-stranded) increases linearly with the %G and the
%C present in the RNA.
TheTmis also dependent on the molar content of protons
in the solution and the formamide percentage in the buffer.
This is summarized in the following equation:
Tm ¼ 81.5 C + 16.6 log[Na+] + 0.41 (%
GC)0.63(% formamide) [9, 10]. The annealing tempera-
ture of nucleotides is considered to be 5C lower than theTm. - The specificity of the antisense probes needs to be checked
using different controls. The best are hybridizations with the
sense probe or without probe, and preincubation with RNaseA
(10μg/mL1 in PBS at 37 C for 15 min) before the
hybridization procedure.
In case of nonspecific labeling and/or high background,
you can assess which steps create it by omitting the primary or
secondary antibodies (if used) and skip the double labeling.
Also use sections from other species where the mRNA is not
expressed. - Of note, the anti-DIG antibody used in this protocol is raised
in sheep, and cannot be directly recognized by PAG and neces-
sitates the need of a bridging rabbit anti sheep antibody that
can lead to the clustering of the PAG. This could be an advan-
tage for less abundant transcripts.
Acknowledgments
I thank Dr. Bram Herpers (Leiden, NL) in collaboration with Ilan
Davis and his group (Oxford, UK), and Tim Weil (Cambridge, UK)
for developing and using this technique.mRNA Detection by EM 185