Adult C57B/6 mice (Jackson Labs) were transcardially perfused with 4% PFA and 0.5% glutaraldehyde (EMS) in 100 mM phosphate buffer (PB). Brains were post-fixed at 4°C overnight, and 50 μm coronal sections were cut on a Leica VT1000 S vibratome. Pre-embedding IHC was performed using WFA (Sigma L1516), amplified with Vectastain Elite ABC kit (Vector Laboratories), and developed with DAB23 (link). Sections were postfixed in 1% osmium tetroxide for 30 min and then embedded in Durcupan ACM epoxy resin (Fluka, Sigma-Aldrich)51 . For reconstruction of WFA-labeled neurons, we cut ~200 serial semithin (1.5 μm) sections on an Ultracut UC-6 ultramictrotome. Selected semithin sections were glued to resin blocks and detached from glass slides by repeated freeze-thaw. Ultrathin sections (60–80 nm) were then cut and placed on Formvar-coated single-slot grids, stained with lead citrate, and examined at 80 kV on a FEI Tecnai G2 Spirit transmission electron microscope equipped with a Morada CCD digital camera (Olympus).
Tecnai g2 spirit transmission
Manufactured by Olympus
The Tecnai G2 Spirit is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of nanoscale structures. It features a LaB6 electron source, a high-resolution objective lens, and advanced image processing capabilities. The Tecnai G2 Spirit is capable of producing detailed, high-quality images of a wide range of materials, including biological samples, polymers, and inorganic compounds.
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2 protocols using tecnai g2 spirit transmission
1
Ultrastructural Analysis of WFA-Labeled Neurons
2
Ultrastructural Analysis of WFA-Labeled Neurons
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Adult C57B/6 mice (Jackson Labs) were transcardially perfused with 4% PFA and 0.5% glutaraldehyde (EMS) in 100 mM phosphate buffer (PB). Brains were post-fixed at 4°C overnight, and 50 μm coronal sections were cut on a Leica VT1000 S vibratome. Pre-embedding IHC was performed using WFA (Sigma L1516), amplified with Vectastain Elite ABC kit (Vector Laboratories), and developed with DAB23 (link). Sections were postfixed in 1% osmium tetroxide for 30 min and then embedded in Durcupan ACM epoxy resin (Fluka, Sigma-Aldrich)51 . For reconstruction of WFA-labeled neurons, we cut ~200 serial semithin (1.5 μm) sections on an Ultracut UC-6 ultramictrotome. Selected semithin sections were glued to resin blocks and detached from glass slides by repeated freeze-thaw. Ultrathin sections (60–80 nm) were then cut and placed on Formvar-coated single-slot grids, stained with lead citrate, and examined at 80 kV on a FEI Tecnai G2 Spirit transmission electron microscope equipped with a Morada CCD digital camera (Olympus).
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