Sort-purified lin−CD90.2+KLRG1−SI LC3s from Flt3tg were cultured 48 h with or without 20 ng per mL IL-23 in the presence or absence of rapamycin (10 or 100 nM). After stimulation, the cells were fixed in PBS containing 3.75% paraformaldehyde for 30 min at room temperature and washed twice with 1% BSA in PBS. Then, cells were permeabilized for 30 min with 0.1% Triton X100 in 1% BSA/PBS and washed once. After blocking with 1% goat serum in 1% BSA/PBS, cells were incubated overnight with primary mouse α-tubulin (Sigma-Aldrich) and rabbit α-p-S6 Ser235/236 (Cell Signaling Technology) Abs. Finally, cells were washed three times with 0.1% Triton X100 in 1% BSA/PBS, incubated for 1 h with secondary goat α-mouse IgG IRDye680 (LI-COR Biosciences) and goat α-rabbit IgG IRDye800 (LI-COR Biosciences) Abs and washed five times with 0.1% Triton X100 in 1% BSA/PBS. Before scanning the cells with the Odyssey CLx Infrared Scanning System (LI-COR Biosciences), all supernatant was removed. Image Studio Software (LI-COR) using Odyssey CLx Infrared Scanning System (LI-COR Biosciences) was used for data collection.
Odyssey clx infrared scanning system
Manufactured by LI COR
Sourced in United States
The Odyssey CLx infrared scanning system is a versatile laboratory instrument designed for the detection and quantification of fluorescent and near-infrared (NIR) labeled molecules. The system utilizes high-sensitivity scanning technology to capture images of gel-based and membrane-based assays, providing accurate and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Image studio software, by LI COR (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Image studio lite ver 3, by LI COR (2 mentions)
α syn, by Santa Cruz Biotechnology (1 mentions)
Rainbow molecular weight marker, by Cytiva (1 mentions)
6 protocols using odyssey clx infrared scanning system
1
IL-23 and Rapamycin Modulate S6 Phosphorylation
2
α-Synuclein Levels in Rat Brain Regions
3
Protein extraction and analysis from mouse tissues
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For brain and retina, freshly excised mouse tissues were flash-frozen in liquid nitrogen and homogenized by grinding using mortar and pestle in liquid nitrogen, then resuspended in SDS sample buffer, fractionated by SDS-PAGE and transferred onto nitrocellulose membranes for Western blotting16 (link). For transfected cells, after washing with PBS on ice, cells were collected into Eppendorf tubes and centrifuged at 17000g for 5 min, then PBS was removed and the pellets were weighted and resuspended in 1:10 (w/v) of 2 SDS buffer, heated to 95 C for 15 min, then centrifuged at 17000g for 15 min. The supernatants were diluted with 1 SDS buffer and loaded onto 12% SDS-PAGE for Western blot. Protein bands were detected using the Odyssey CLx infrared scanning system and quantified with Image Studio Lite Ver3.1 (Li Cor, Lincoln, NE, USA). Signal intensity was normalized to GAPDH signal for comparison of protein levels between samples.
4
Transfected Cell Protein Analysis
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For transfected cells, after washing with PBS on ice, cells were collected into Eppendorf tubes and centrifuged at 17000 g for 5 min, then PBS was removed and the pellets were weighted and resuspended with 1:10 (w/v) of 2xSDS buffer, heated to 100 °C for 15 min, then centrifuged at 17000 g for 15 min. The supernatant was diluted with 1xSDS buffer and loaded onto 12% SDS- PAGE for Western blot. For testing for the presence of extra aggregated proteins, cells were collected as described above and resuspended in lysis buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.2% Triton X-100, plus protease inhibitor cocktail) and kept on ice for 15 min to facilitate cell lysis. Cell lysates were then centrifuged at 50,000 g, and the pellets after this spin were dissolved in SDS sample buffer for Western blot analysis with Syn303.
Fractionation of the brain lysates into soluble and insoluble fractions by sequential Triton X-100 lysis was performed as described in ref. 35 .
Protein bands were detected using the Odyssey CLx infrared scanning system and quantified with Image Studio Lite Ver3.1(Li Cor, Lincoln, Nebraska). Signal intensity was normalized by quantitative (q)PCR to evaluate the transfection efficiency and by loading controls tubulin and GAPDH for comparison of protein levels between samples.
Fractionation of the brain lysates into soluble and insoluble fractions by sequential Triton X-100 lysis was performed as described in ref. 35 .
Protein bands were detected using the Odyssey CLx infrared scanning system and quantified with Image Studio Lite Ver3.1(Li Cor, Lincoln, Nebraska). Signal intensity was normalized by quantitative (q)PCR to evaluate the transfection efficiency and by loading controls tubulin and GAPDH for comparison of protein levels between samples.
5
Verifying PFF Formation in α-Syn Solutions
6
Western Blot Analysis of Myelin and AMPA Proteins
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