Neo micro cover glass

For GFP observation, plants that were to be directly compared were grown side-by-side at the same density per pot to minimize potential micro-environmental differences in the growth chamber. Petals were harvested in PCR tubes containing 3.3% (w/v) paraformaldehyde (Nacalai tesque) and 0.16 mg/mL propidium iodide (Sigma) dissolved into 1× phosphate-buffered saline (PBS) (Takara) and incubated overnight at 4 °C. The paraformaldehyde solution was replaced with Clear See (FUJIFILM Wako Chemicals) and maintained for about 1 week until the tissues became transparent. The resulting tissues were placed onto a NEO micro slide glass (MATSUNAMI), mounted on a NEO micro cover glass (MATSUNAMI) with one or two drops of water or Clear See, and observed immediately with a confocal laser scanning microscope (SP8 or Stellaris 5: Leica). The same offset and gain settings were used for all plants for which signal was directly compared. More than 10 petals for each genotype and each position were observed, and representative images are shown.

A series of paraffin sections at 5 µm, 15 µm, 30 µm, and 50 µm thickness were cut and mounted onto microscope slides as described above. Deparaffinized and rehydrated sections were stained with Mayer’s hematoxylin for 3 min and exposed to running tap water at least for 3 hours to develop the colour. Next, the sections were stained with eosin diluted in 60% ethanol for 3 min, then rinsed in a graded series of 80%, 90%, and 100% ethanol for dehydration. After clearance in xylene, the sections were mounted within Malinol (Muto Pure Chemicals, Tokyo, Japan) covered with a NEO Micro cover glass (size 24 × 50 mm, thickness No.1 = 0.13–0.17 mm: Matsunami Glass, Osaka, Japan). After observations under a light microscope (BX51, Olympus, Tokyo, Japan) equipped with a digital camera (DP72, Olympus), the microscope slides were incubated in xylene for 18–24 hours at room temperature to remove the coverslips (Fig. 1f). The sections were rehydrated with a series of 100%, 90%, and 70% ethanol and distilled water (5 min each), then stained with UA and LC as described above.