- Cut ultrathin frozen sections (70–80 nm) at 120 Cinan
ultracryomicrotome [4] and collect them with a drop of
methylcellulose–sucrose on Nickel 50 mesh grids supporting
a carbon-coated formvar film (Fig.2).
3.3 ISH on Grids 1. After washing the excess of MC–sucrose by floating the grids
(sections-side down) on drops of DEPC-treated PBS for
10 min at 37C, post-fix them in 1% GA in PBS for 5 min at
RT or in 4% PFA in PBS for 15 min at 37C(seeNote 6)
Fig. 2Schematics representation of critical steps of the ISH-IEM method. (a) Ultrathin frozen sections of a
Drosophila egg chamber embedded in a gelatin block are cut on a diamond knife in an ultramicrotome. (b) The
sections are collected on a formvar coated EM nickel grid. (c) Grids are incubated in a humid hybridization
chamber in microfuge caps
182 Catherine Rabouille