(2014)

(2014). in living cells. We now demonstrate the successful application of GSDIM to archival human brain tissue sections including from Alzheimer’s disease cases as well as experimental tissue samples from mouse and zebrafish larvae. Presynaptic terminals and microglia and their cell processes were visualized at a resolution beyond diffraction\limited light microscopy, allowing clearer insights into their interactions (1) PBS. Sections were subsequently washed five times for 5 min in 1 PBS made up of 0.3% Verbascoside tween\20 (PBST; pH 7.4). Blocking was performed in 10% NGS (Cat# 16210\072, Thermo Fisher Scientific, Waltham, Massachusetts, United States) diluted in 1 PBST followed by primary antibody incubation (anti\ionized calcium\binding adaptor molecule 1 (Iba1), 1:1,000, Cat# 019\19471, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, RRID:AB_839504; anti\synaptophysin (Syp), 1:200, Cat# M0776, Dako, Denmark, RRID:AB_2199013), and secondary antibody incubation (1:500; Alexa Fluor (AF) 532 goat anti\rabbit, Cat# A32728, RRID:AB_2534076; Alexa Fluor 647 goat anti\mouse, Cat# A11009, RRID:AB_2633277; ThermoFisher Scientific), both in 1% NGS at 4C overnight with gentle agitation. Sections were then washed five times for 5 min in 1 PBS. Previously, sections for GSDIM (imaged according to old gold\standard acquisition settings) were stained as described here but incubated in AF 647 (1:200, donkey anti\rabbit, Cat# A\31573, ThermoFisher Scientific, RRID:AB_2536183) and AF 568 (1:200; goat anti\mouse, Cat# A11004, ThermoFisher Scientific, RRID:AB_2534072) and quenched using 0.1% Sudan Black B (BDH Laboratory Chemical Group, United Kingdom) in 70% ethanol for 4 min, then stored in 1 PBS at 4C until Verbascoside imaging. Further methodological information can be found in the Supporting Information Methods. Table?2 lists all antibodies used here. TABLE 1 Basic clinical data of human cases used in this study test, post hoc ANOVA: fixed effects, omnibus, one\way; RRID:SCR_013726). Groups were controlled for age, sex (which was not disaggregated due to the small group sizes), fixation time, post\mortem index, and brain pH. Spearman was calculated to identify potential correlations between the percentage of co\localized Iba1/Syp pixels and the aforementioned factors. Rabbit polyclonal to LYPD1 A value?