Deconstructing Complexity: Serial Block-Face Electron Microscopic Analysis of the Hippocampal Mossy Fiber Synapse (original) (raw)

“…1851 (Miltenyi Biotec) and subsequently fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at 4°C, followed by 1 h in 2% OsO 4 , 1.5% potassium ferrocyanide in 0.15 M cacodylate, and 20 min at room temperature in 1% thyocarbohydrazide to allow additional osmium staining (55). Material was stained en bloc with 2% uranyl acetate and Walton's lead aspartate for maximum membrane contrast as described (55) before embedding in Durcupan Araldite embedding resin. Gametocytes were distinguished from asexual parasites by the presence of an inner membrane complex in the parasites (34).…”

Section: Discussionmentioning

“…For SBEM, the ONHs of two male C57BL/6J mice (aged 3 mo and 9 mo) were processed with procedures modified from Wilke et al (19), essentially as previously described by Nguyen et al (4). Volumes were processed and analyzed using IMOD software (http://bio3d.…”

Section: Methodsmentioning

“…Selected pieces were processed in 2 ml Eppendorf tubes for embedding as previously described (Wilke et al 2013). In brief, after fixation, specimens were washed extensively with Na-cacodylate buffer, pH 7.4, supplemented with 2 mM CaCl 2 , and subjected to double-osmication comprising incubations with 2 % OsO 4 -1.5 % K 4 [Fe (CN) 6 ] in Na-cacodylate/CaCl 2 buffer for 1 h on ice, 1 % thiocarbohydrazide for 20 min at RT and 2 % OsO 4 for 30 min at RT.…”

Section: Sample Preparation For Sbemmentioning