- For protein detection after mRNA, proceed with double or
triple labeling of the sections, by repeatingsteps 1–4using
appropriate primary antibodies and PAG of different sizes [4].
Rabbit polyclonal antibodies are directly detected by PAG (step
5 ). Mouse and sheep monoclonal antibodies are decorated by a
rabbit anti-mouse and anti-sheep IgGs, respectively, followed
by PAG (step 3a).
3.5 Contrasting 1. Wash the grids in ten drops of dH 2 O for 10 min.
- Contrast the sections by floating the grids for 5–10 min on 2%
UA oxalate. - Pass the grids over one or two drops of UA-MC [6] and leave
them in the third drop for 5–10 min on ice. - Use a wire loop to scoop the grid from the UA-MC, remove
excess UA-MC by dragging the loop with grid over a filter
paper and let the grid dry in air for at least 10 min. - Pinch out the grid from the wire loop and store the grid in a
grid box. - Visualize the results by using a transmission electron micro-
scope at 80 kV.
4 Notes
- Most of the reagents involved in the procedure are toxic. They
need to be manipulated in the fume hood and with gloves. - For easier comparison, the two ovaries from the same female
can be fixed differently. Fixation is the key step in this protocol.
The resulting morphology and labeling depend on the fixative
used. Furthermore, fixation should be performed at RT, not at
4 C and the fixative diluted in phosphate buffer pH 7.4, not
PBS. The ovaries have to be fixed immediately upon excision
and should not be stored in the Ringer buffer. Other tissues can
also be used but their fixation might need to be done by
perfusion (not only by immersion) to keep an optimal ultra-
structure, for instance for rat liver or brain [5] - Most commercial gelatins give a precipitate (probably calcium
phosphate) when prepared in phosphate buffer, but they can be
prepared in different buffers (e.g., Tris–HCl or Tris-buffered
saline). The concentration should be adjusted to the desired
stiffness of the resulting pellets [4]. - Nickel grids: 50 mesh grids are optimal for viewing sections of
large cells such as the Drosophila oocytes. For small cells,
smaller meshes are adequate and provide a stronger support.
However, only nickel grids are suitable for the ISH procedure
184 Catherine Rabouille