10.2 How to observe the cellular localization of a fluorescent protein

Takashi Murata and Yoshikatsu Sato

For analyzing cellular localization of a protein fused to a fluorescent protein, we need to observe protonemata without damage under good optical conditions. For this purpose, protonemata are cultured along the glass surface of a glass-bottomed dish (IWAKI 3910-039). The thickness of the agar medium is minimized to obtain good optical conditions.　 Additionally, having a large mass of agar medium connected with the thin layer of the medium, in which the protonemata grow, allows continuous nourishment and growth of the gametophyte.

Methods

1. Pour 2 ml of BCD medium onto the plastic part of the glass-bottom dish and allow to set for 30 min.

2. Pour 65 µl of BCD medium on the glass central region of the dish.

3. A small colony of protonemata is inoculated into the glass-bottom dish with medium, and then overlaid with cellophane. After 3 or 4 days of culture, protonemata grow parallel to the bottom of the dish.

Observation should be performed by an inverted microscope.