A method for cleaning freeze-fracture replicas is elaborated which avoids their rolling up and fragmentating during successive steps. Immediately after freeze-fracturing, the replicated tissue is slowly thawed from 77 K to room temperature on solid methanol. Safe passage through the subsequent cleaning steps is facilitated by using solutions of gradually increasing surface tension. After at least 24 h of fixation in methanol, the tissue is digested in half-strength bleach containing 5% ethanol. Following further cleaning in full-strength bleach, the remaining organic material attached to the replica is dissolved overnight at room temperature in 50% saturated sodium hydroxide.
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http://dx.doi.org/10.1111/j.1365-2818.1985.tb02576.x | DOI Listing |
J Cell Biol
January 2025
Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a phospholipid essential for plasma membrane functions, but its two-dimensional distribution is not clear. Here, we compared the result of sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) of quick-frozen cells with the actual PtdIns(4,5)P2 content and the results obtained by fluorescence biosensor and by labeling of chemically-fixed membranes. In yeast, enrichment of PtdIns(4,5)P2 in the membrane compartment of Can1 (MCC)/eisosome, especially in the curved MCC/eisosome, was evident by SDS-FRL, but not by fluorescence biosensor, GFP-PLC1δ-PH.
View Article and Find Full Text PDFBiol Res
August 2024
Synaptic Structure Laboratory, Departamento de Ciencias Médicas, Facultad de Medicina, Instituto de Biomedicina de la UCLM (IB-UCLM), Universidad Castilla-La Mancha, Campus Biosanitario, C/ Almansa 14, Albacete, 02008, Spain.
Synaptic dysfunction is an early feature in Alzheimer's disease (AD) pathogenesis and a major morphological correlate of memory deficits. Given the main synaptic location of N-methyl-D-aspartate receptors (NMDARs), their dysregulation has been implicated in these pathological effects. Here, to detect possible alterations in the expression and synaptic localisation of the GluN1 subunit in the brain of amyloidogenic APP/PS1 mice, we employed histoblot and SDS-digested freeze-fracture replica labelling (SDS-FRL) techniques.
View Article and Find Full Text PDFBrain Pathol
June 2024
Departamento de Ciencias Médicas, Facultad de Medicina, Synaptic Structure Laboratory, Instituto de Biomedicina de la UCLM (IB-UCLM), Universidad Castilla-La Mancha, Albacete, Spain.
Voltage-gated Ca2.1 (P/Q-type) Ca channels play a crucial role in regulating neurotransmitter release, thus contributing to synaptic plasticity and to processes such as learning and memory. Despite their recognized importance in neural function, there is limited information on their potential involvement in neurodegenerative conditions such as Alzheimer's disease (AD).
View Article and Find Full Text PDFNeuron
March 2024
Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria. Electronic address:
The coupling between Ca channels and release sensors is a key factor defining the signaling properties of a synapse. However, the coupling nanotopography at many synapses remains unknown, and it is unclear how it changes during development. To address these questions, we examined coupling at the cerebellar inhibitory basket cell (BC)-Purkinje cell (PC) synapse.
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