In situ microwave fixation provides an instantaneous snapshot of the brain metabolome

Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EVISF) reveals sex‐dependent changes in microglial EV proteome in response to Aβ pathology

“…While this includes the study of brain‐EVs from humans, rodent models, and nonhuman primates, postmortem analyses have caveats. The postmortem interval can alter RNA expression and integrity (Ferreira et al., 2018 ), shift brain metabolism (Juras et al., 2023 ), and accelerate protein degradation (Blair et al., 2016 ). Thus, postmortem tissue changes may affect brain‐derived EVs by causing artifactual changes in the EV cargo that would be absent if isolated from a live brain.…”

Mass spectrometry imaging highlights dynamic patterns of lipid co‐expression with Aβ plaques in mouse and human brains

“…The unexpected differences in lipid composition between h‐Aβ and WT mouse brains may be influenced by factors such as diet or environment, warranting additional studies using animals raised together to confirm the observed differences. Finally, we used were from snap frozen tissues as described in several previous studies (Kaya et al., 2017, 2018a; Michno et al., 2018, 2022), although some reports have emphasized the need for more rapid fixation using high‐energy microwave‐based fixation in situ to arrest metabolism more rapidly (Juras et al., 2023; Murphy, 2010). Instead, we worked to mitigate any effects of residual enzymatic activity for the conclusions here by careful standardization of the protocol across all groups being prepared, rapid vacuum desiccation after sample freezing, and using methanol as a solvent for the DESI MSI, so we believe that relative differences are meaningful.…”

“…Mice were maintained on a 12/12 h light/dark cycle in a group with sex-matched littermates (maximum 5 mice per cage) in individually ventilated cage racks with drinking nozzles attached to the individual cage to allow ad libitum access to reverse osmosis-purified water and food (standard laboratory diet of irradiated 1324 pellets, Altromin Spezialfutter GmbH, Lage, Germany). While an alternative protocol for rapid in situ fixation using high energy head-focused microwave prior snap freezing has been proposed (Juras et al, 2023), we used the more frequently reported approach for MSI studies of snap freezing (Kaya et al, 2017(Kaya et al, , 2018a(Kaya et al, , 2020(Kaya et al, , 2023. Mice that were 2.5, 7, or 12 months old and did not undergo any other procedures received an intraperitoneal injection of sodium pentobarbital (200 mg/kg, Jurox).…”

The multifaceted roles of the brain glycogen

“…In fact, research on glycogen extraction methods and degradation time suggests that up to 95% of glycogen may be degraded during improper experimental and extraction conditions (Cruz & Dienel, 2002;Dienel, 2020;Hsu et al, 2021). The implementation of focused microwave fixation in situ has provided a significant advantage for the accurate detection of physiological glycogen compared with other approaches (Juras et al, 2023;Oe et al, 2016).…”