Afsii

Plp^−/Y mice (Klugmann et al., 1997 ) kept on a C57Bl/6N background and wild type littermates were sacrificed by cervical dislocation at postnatal day 75 (P75). The optic nerves were dissected, cut and high-pressure frozen (Weil et al., 2019 ) using 20% Polyvinylpyrrolidone (PVP) as a filler and the HPM100 high-pressure freezer (Leica, Vienna, Austria). An automatic freeze substitution unit (Leica, AFSII) was used to perform the freeze substitution. 0.1% tannic acid in acetone was incubated at −90° C for 100 h followed by three acetone rinses for 30 min each. 2% OsO_4, 0.1% uranyl acetate in acetone was used for contrasting the samples (7 h at −90 °C). The temperature was automatically raised to −20 °C within 14 h, kept there for 16 h and raised to 4 °C within 2.5 h. OsO₄ was removed by washing four times with acetone for 30 min. The nerves were then rinsed with acetone for 1 h at room temperature. The samples were incubated with increasing concentrations of resin (2:1, 1:1, 1:2) for 2 h each and left in 90% Durcupan over night without component D. The next day the samples were incubated with 100% Durcupan (all components) for 4.5 h and polymerized for 48 h at 60 °C.

Macrophage samples were high-pressure frozen in an Abra HPM-010 machine and freeze-substituted in 99% acetone, 0.1% uranyl acetate, 1% distilled water (dH₂O) in a Leica AFSII automatic freeze-substitution device. Freeze substitution was performed at −90°C for 72 h and then ramped to −50°C over 24 h, at which point cells were placed in 4-h-graded steps of 25%, 50%, 75%, 100% HM-20 in acetone. HM-20 was polymerized with 360-nm light for 48 h at −50°C and an additional 24 h at room temperature. Polymerized blocks trimmed to regions of interest were cut at 60- to 80-nm thicknesses and immunologically probed with IgG-purified type 23F rabbit serum. Protein A conjugated to 15-nm colloidal gold beads was used to secondarily detect the presence of antibody-antigen complexes. Imaging was performed on a JEOL 1010 transmission electron microscope (TEM) operating at 80 keV.