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Afs freeze substitution instrument

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The AFS freeze-substitution instrument is a specialized piece of laboratory equipment used for the preparation of biological samples for electron microscopy. The core function of this instrument is to carefully control the temperature and chemical environment during the process of freeze-substitution, which involves the replacement of water in frozen samples with an organic solvent.

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Em cpc, by Leica (2 mentions)

4 protocols using afs freeze substitution instrument

1

Postembedding Immunogold Labeling of Cerebellum

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Postembedding immunogold labeling of the cerebellum was performed using established materials and methods [8 (link)-10 (link)]. Fixed tissue sections from two P37 Sprague-Dawley rats were cryoprotected and frozen in a Leica CPC using liquid propane, then embedded in Lowicryl HM-20 resin in a Leica AFS freeze-substitution instrument. After blocking with normal goat serum, sections were incubated with the rabbit RB a-1b and mono a-1a antibodies. Following incubation with 5 and 15 nm gold-conjugated secondary antibodies, sections were stained with uranyl acetate and lead citrate.
Techlovská Š., Chambers J.N., Dvořáková M., Petralia R.S., Wang Y.X., Hájková A., Nová A., Franková D., Prezeau L, & Blahos J. (2014). Metabotropic Glutamate Receptor 1 Splice Variants mGluR1a and mGluR1b Combine in mGluR1a/b Dimers in vivo. Neuropharmacology, 86, 329-336.
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2

Double-Immunogold Labeling of Rat Brain

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Animals used for postembedding, double-immunogold localization were prepared as described previously (Petralia and Wang, 2021 (link)). Briefly, two male, adult Sprague Dawley rats were perfused with phosphate buffer, followed by perfusion with 4% paraformaldehyde + 0.5% glutaraldehyde in phosphate buffer, and then the brains were vibratomed, cryoprotected in glycerol overnight, frozen in a Leica EM CPC (Vienna, Austria), and embedded in Lowicryl HM-20 resin in a Leica AFS freeze-substitution instrument. Thin sections were incubated in 0.1% sodium borohydride + 50 mM glycine in Tris-buffered saline plus 0.1% Triton X-100 (TBST), then in 10% normal goat serum (NGS) in TBST, and then with 2 primary antibodies together in 1% NGS/TBST (overnight); then they were incubated with the 2 immunogold-conjugated secondary antibodies (5+15 nm; Ted Pella, Redding, CA, USA) in 1% NGS in TBST with 0.5% polyethylene glycol (20,000 MW) and stained with uranyl acetate and lead citrate. Controls on sections from the same two rats, labeled with the 2 secondary antibodies but without the primary antibodies, showed only rare gold and no colocalization.
Murphy J.G., Gutzmann J.J., Lin L., Hu J., Petralia R.S., Wang Y.X, & Hoffman D.A. (2022). R-type voltage-gated Ca2+ channels mediate A-type K+ current regulation of synaptic input in hippocampal dendrites. Cell reports, 38(3), 110264.
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3

Postembedding Immunogold Labeling Protocol

Cited 1 time
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Postembedding immunogold labeling was performed as described previously (Petralia and Wenthold 1999 (link); Petralia et al. 2010 (link); Yao et al. 2015 (link)). Briefly, following cryoprotection and embedding, rat brain sections were processed and embedded in Lowicryl HM-20 resin using a Leica AFS freeze-substitution instrument. After blocking, the sections were incubated with primary antibody followed by 10-nm gold-conjugated secondary antibody. Following thorough washes, the sections were stained with uranyl acetate and lead citrate. All electron micrographs were stored in their original formats. For micrographs presented for figures, their levels, brightness and contrast were minimally and evenly adjusted in Adobe Photoshop. Control sections omitting the primary antibody showed only rare gold labeling.
Williamson J., Petralia R.S., Wang Y.X., Mattson M.P, & Yao P.J. (2017). Purine Biosynthesis Enzymes in Hippocampal Neurons. Neuromolecular medicine, 19(4), 518-524.
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4

Double-Immunogold Labeling of Rat Brain

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Animals used for postembedding, double-immunogold localization were prepared as described previously (Petralia and Wang, 2021 (link)). Briefly, two male, adult Sprague Dawley rats were perfused with phosphate buffer, followed by perfusion with 4% paraformaldehyde + 0.5% glutaraldehyde in phosphate buffer, and then the brains were vibratomed, cryoprotected in glycerol overnight, frozen in a Leica EM CPC (Vienna, Austria), and embedded in Lowicryl HM-20 resin in a Leica AFS freeze-substitution instrument. Thin sections were incubated in 0.1% sodium borohydride + 50 mM glycine in Tris-buffered saline plus 0.1% Triton X-100 (TBST), then in 10% normal goat serum (NGS) in TBST, and then with 2 primary antibodies together in 1% NGS/TBST (overnight); then they were incubated with the 2 immunogold-conjugated secondary antibodies (5+15 nm; Ted Pella, Redding, CA, USA) in 1% NGS in TBST with 0.5% polyethylene glycol (20,000 MW) and stained with uranyl acetate and lead citrate. Controls on sections from the same two rats, labeled with the 2 secondary antibodies but without the primary antibodies, showed only rare gold and no colocalization.
Wood G.E., Dutro S.M, & Totten P.A. (1999). Target Cell Range of Haemophilus ducreyi Hemolysin and Its Involvement in Invasion of Human Epithelial Cells. Infection and Immunity, 67(8), 3740-3749.
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