ホスファターゼ酵素組織化学

Acid phosphatase activity was detected using TEM with a cerium-based method (Dreyer et al., 2008) with some modifications.

1. Root fragments (5–10 mM) were pre-fixed with 2.5% glutaraldehyde in 100mM cacodylate buffer (pH 7.0) for 2h on ice.
2. The fragments were washed twice with cacodylate buffer for 30min. Roots were further cut into 0.5 mM fragments.
3. The fragments were sequentially incubated in a pre-incubation buffer \[2mM CeCl3 in 100mM acetate buffer (pH 4.6)] for 1 h, a reaction buffer (1 mM β-glycerophosphate and 2 mM CeCl3 in acetate) for 30min, a pre-incubation buffer for 15min, and an acetate buffer for 15min at 37°C.
4. Samples were post-fixed with 1% OsO4 in cacodylate buffer for 2h on ice, followed by washing with DW thrice for 5min.
5. Roots were dehydrated with an ethanol series (20, 50, 70, 90, and 95% once and 100% thrice) for 20min each at 4°C
6. Incubate twice with propylene oxide for 5min at room temperature.
7. The samples were infiltrated with Spurr’s resin:propylene oxide (1:2, 1:1, 2:1, 3:1, and 4:1) for 1h in each step and thrice with pure resin for 3 h
8. Polymerized at 70°C for 12 h.
9. Ultrathin sections on copper grids were stained with gadolinium acetate (Nakakoshi et al., 2011) and lead citrate
10. Observed using a TEM at an accelerating voltage of 80 kV.
11. Controls were prepared by incubating sections without β-glycerophosphate in the reaction buffer and without 2mM CeCl3 in the pre-incubation and reaction buffers.