Preparation for SEM (LEO 435 VP, Leica, Wiesbaden, Germany) involved successive dehydration in methanol (30%, 70%, 99,9%, each step for 2–3 days) or rapid methanol fixation32 (link), critical-point drying (LPD 030, Bal-Tec/Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany) and gold-sputtering (Sputter Coater 108 auto, Cressington Scientific Instruments Ltd., Watford, England UK).
Preparation for LM involved fixation (90 parts 50% isopropanol and 10 parts 99.5% glycerine) and infiltration with Technovit 7100 (Heraeus Kulzer GmbH, Hanau, Germany). The material was sectioned on a sliding microtome at 10 µm thickness. Sections were stained with 10% aqueous toluidine blue (Chroma-Incorporation, Stuttgart, Germany) and alternatingly rinsed with deionized water and 100% isopropanol. The microscopy slides were sealed with Entellan (Merck, Darmstadt, Germany). A BX61 automated light microscope (Olympus Life Science Corp., Hamburg, Germany) equipped with a DP71 digital camera and the software Cell-P 2.8 (Olympus Soft Imaging Solutions) were used.
Preparation for LM involved fixation (90 parts 50% isopropanol and 10 parts 99.5% glycerine) and infiltration with Technovit 7100 (Heraeus Kulzer GmbH, Hanau, Germany). The material was sectioned on a sliding microtome at 10 µm thickness. Sections were stained with 10% aqueous toluidine blue (Chroma-Incorporation, Stuttgart, Germany) and alternatingly rinsed with deionized water and 100% isopropanol. The microscopy slides were sealed with Entellan (Merck, Darmstadt, Germany). A BX61 automated light microscope (Olympus Life Science Corp., Hamburg, Germany) equipped with a DP71 digital camera and the software Cell-P 2.8 (Olympus Soft Imaging Solutions) were used.