Jem 100 sx microscope

A Leica Ultracut UCT ultramicrotome was used to prepare ultrathin sections (50 nm). They were blocked in 1% BSA (Aurion, Wageningen, the Netherlands) in PBS buffer for 15 min and then incubated in primary antibodies in a 1:10 dilution overnight at 4 °C. Followed by washes in PBS buffer (6 × 5 min) and incubated with the goat antirat secondary antibody conjugated with 10 nm colloidal gold (Sigma Aldrich, Poland) in a 1:50 dilution for 2 h, followed by washing in PBS buffer and distilled water. Negative controls were created by omitting the primary antibody step (Figure S1C). Lead citrate (Microshop, PIK Instruments Sp. z o.o., Piaseczno, Poland) and URANYLess (Microshop, PIK Instruments Sp. z o.o., Piaseczno, Poland) were added as contrasting agents. The cells were visualized using a Jeol JEM 100 SX microscope (JEOL, Tokyo, Japan) at 80 kV in the Department of Cell Biology and Imaging, Institute of Zoology, Jagiellonian University in Kraków or a Hitachi UHR FE-SEM SU 8010 microscope at 25 kV, housed at the University of Silesia in Katowice.

The traps were also examined using transmission electron microscopy (TEM), as follows: Fragments of the traps were fixed in a mixture of 2.5% glutaraldehyde with 2.5% formaldehyde in a 0.05 M cacodylate buffer (Sigma-Aldrich, Sigma-Aldrich Sp. z o.o. Poznań, Poland; pH 7.2) overnight or for several days, washed three times in a 0.1 M sodiuM cacodylate buffer, and post-fixed in a 1% osmium tetroxide solution at room temperature for 1.5 h. This was followed by dehydration using a graded ethanol series, infiltration, and embedding using an epoxy embedding medium kit (Fluka). Following polymerization at 60 °C, sections were cut at 70 nm for the transmission electron microscopy (TEM) using a Leica ultracut UCT ultramicrotome, stained with uranyl acetate and lead citrate [59 (link)] and visualized using a Jeol JEM 100 SX microscope (JEOL, Tokyo, Japan) at 80 kV in the Department of Cell Biology and Imaging, Institute of Zoology, Jagiellonian University in Kraków or with a Hitachi UHR FE-SEM SU 8010 microscope at 25 kV, which is housed at the University of Silesia in Katowice.