Removed brains were mounted and cut into coronal slices (300 μm) on a vibrating blade microtome (Leica) in cold HBSS supplemented with 100 U/ml penicillin/streptomycin, 10 mM HEPES, and 4.5 mM sodium bicarbonate. The SCN slices were dissected out and transferred onto a PTFE membrane insert (Millipore) in 35 mm culture dishes with 1.2 ml of DMEM (D5030, Sigma) supplemented with 3.5 g/L D-glucose, 2 mM Glutamax (Gibco), 10 mM HEPES, 25 U/ml penicillin/streptomycin, 2% B-27 Plus (Gibco), and 0.1 mM D-Luciferin sodium salt (Tocris). The SCN slice position was adjusted to the center of the dish and 1.5 μl AAV (pAAV1-Syn-ChrimsonR-tdT, Addgene) (Klapoetke et al., 2014 (link)) was placed directly onto the SCN slice. The culture dishes were then sealed with an optically clear PCR plate film (Bio-Rad) and maintained in a non-humidified incubator at 36.8 °C for about 10 days. The opsin expression was checked after about 10 days of viral transduction by imaging tdT fluorescence.
Ptfe membrane insert
Manufactured by Merck Group
The PTFE membrane insert is a laboratory equipment component designed to facilitate fluid filtration and separation processes. It features a membrane made of polytetrafluoroethylene (PTFE), a versatile and chemically resistant material. The core function of this product is to provide a durable and efficient barrier for the separation of solid particles, microorganisms, or other contaminants from liquids or gases, enabling users to achieve precise and reliable results in their laboratory experiments or industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
B 27 plus, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Vibrating blade microtome, by Leica (2 mentions)
Paav1 syn chrimsonr tdt, by Addgene (2 mentions)
Optically clear pcr plate film, by Bio-Rad (2 mentions)
2 protocols using ptfe membrane insert
1
Preparation of SCN Slices for Optogenetics
2
Optogenetic Manipulation of SCN Slices
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Removed brains were mounted and cut into coronal slices (300μm) on a vibrating blade microtome (Leica) in cold HBSS supplemented with 100 U/ml penicillin/streptomycin, 10 mM HEPES, and 4.5 mM sodium bicarbonate. The SCN slices were dissected out and transferred onto a PTFE membrane insert (Millipore) in 35-mm culture dishes with 1.2 ml of DMEM (D5030, Sigma) supplemented with 3.5 g/L D-glucose, 0.2mM Glutamax (Gibco), 10 mM HEPES, 25 U/ml penicillin/streptomycin, 2% B-27 Plus (Gibco), and 0.1 mM D-Luciferin sodium salt (Tocris).
The SCN slice position was adjusted to the center of the dish and 1.5μl AAV (pAAV1-Syn-ChrimsonR-tdT, Addgene) was placed directly onto the SCN slice. The culture dishes were then sealed with an optically clear PCR plate film (Bio-Rad) and maintained in a non-humidified incubator at 36.8 °C for about 10 days. pAAV1-Syn-ChrimsonR-tdT was a gift from Edward Boyden (Addgene plasmid # 59171) 2 . The opsin expression was checked after about 10 days of viral transduction by imaging tdT fluorescence.
The SCN slice position was adjusted to the center of the dish and 1.5μl AAV (pAAV1-Syn-ChrimsonR-tdT, Addgene) was placed directly onto the SCN slice. The culture dishes were then sealed with an optically clear PCR plate film (Bio-Rad) and maintained in a non-humidified incubator at 36.8 °C for about 10 days. pAAV1-Syn-ChrimsonR-tdT was a gift from Edward Boyden (Addgene plasmid # 59171) 2 . The opsin expression was checked after about 10 days of viral transduction by imaging tdT fluorescence.
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