Durcupan acm resin

50 μm thick tissue slices [70 (link)] were rinsed in 1% sodium borohydride in PB for 15 min to eliminate unbound aldehydes. The primary antibodies used for labelling included a polyclonal primary POMC (Invitrogen PA5-18368, 1:1500, incubated overnight) and AgRP antibodies (Invitrogen PA5-78739, 1:200, incubated overnight). As a secondary antibody a biotinylated polyclonal goat anti-rabbit antibody was applied (Invitrogen 65–6140, 1:200, incubated for 1 hr.). Finally, ABC kits (avidin-biotin complex kits, # Pk.4001) and DAB (diaminobenzidine, CAS: 91-95-2) were used for development.
For electron microscopic analysis, sections were osmicated (1% OsO₄ in PB) for 30 min, dehydrated through increasing ethanol concentrations, and embedded into Durcupan ACM resin (Sigma-Aldrich, 44610). Following embedding, ultrathin sections with a thickness of 50 nm were cut for precise examination, (see above the localization of the area in question).
The prepared sections were examined with a JEOL-1011 TEM (JEOL USA, Peabody, MA, USA). The calibrated electron micrographs were analyzed using the NIH Image J (ver. 1.52n) software. The measured parameters included the occurrence (number per area) and size of mitochondria, matrix/entire mitochondrion ratio [71 (link)].

For 3D SBF-SEM analysis the samples were processed as described in Beike et al. (2019 (link)) based on Deerinck et al. (2010b ), in brief: After fixation (0.15 M HEPES buffer with 1.5% glutaraldehyde and 1.5% paraformaldehyde, pH 7.35) small blocks of the samples (edge length 1–2 mm) were stained en bloc applying a rOTO protocol (rOTO: reduced osmium tetroxide “thiocarbohydrazide” osmium tetroxide) with uranyl acetate and lead aspartate to obtain enough contrast and conductivity of the biological structures for SEM imaging. The samples were dehydrated in an ascending acetone series and embedded in Durcupan™ ACM resin (Sigma-Aldrich, St. Louis, USA). To prepare the polymerized resin blocks for SBF-SEM, the embedded samples were roughly trimmed, mounted with conductive epoxy glue (Chemtronics, CircuitWorks, Kennesaw, USA) on an aluminum specimen pin (Gatan, Pleasanton, CA, USA) and precisely retrimmed with glass knives. This was followed by sputter coating of the samples with a 20 nm gold layer.

Using an immunogold immunohistochemistry protocol tissues were prepared for TEM [38 (link),39 (link),40 (link)]. The fixation of the samples was carried out with 4% paraformaldehyde in PBS, after which samples were washed in PBS. Samples were cut with a vibratome (Vibratome Series 1000, Pelco 101, Ted Pella, Inc., Redding, CA, USA) into 20 µm thick sections. After washing in PBS, sections were incubated first in 50% ethanol for permeabilization and then in primary antibody for 48 h at +4 °C: rabbit anti-Cx37/GJA4 (1:100, ab181701, Abcam, Cambridge, UK); rabbit anti-Cx40/GJA5 (1:100, ab213688, Abcam, Cambridge, UK); and goat anti-Cx43/GJA1 (1:300, ab87645, Abcam, Cambridge, UK). Next, sections were rinsed in PBS after which overnight incubation followed with gold-conjugated donkey anti-rabbit or anti-goat secondary antibody (1:1000, 711-205-152 and 705-185-147, both from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The size of the gold particles used was 12 nm for anti-rabbit and 4 nm for anti-goat antibodies. On the next day, sections were rinsed in PBS, post-fixed in 1% osmium tetroxide (1 h), and then dehydrated in ethanol and embedded in Durcupan ACM resin (Sigma-Aldrich Inc., St. Louis, Missouri, USA). The sections were observed with a transmission electron microscope (JEM JEOL 1400, Jeol Ltd., Tokyo, Japan).