Proteoglycans labelled with [35S]-SO4 were prepared for SDS-PAGE by isolation through DEAE-Sephacel anionic exchange mini columns. Samples were added to pre-equilibrated columns and then washed extensively with low salt buffer (8M urea, 0.25M NaCl, 2mM disodium EDTA, 0.5% Triton X-100). Proteoglycans were eluted with high salt buffer (8M urea, 3M NaCl, 2mM disodium EDTA 0.5% Triton X-100) and fractions containing the highest number of [35S]-SO4 cpm were pooled. Equal counts of proteoglycans were precipitated by ethanol solution (1.3% potassium acetate in 95% ethanol) and chondroitin sulphate then added to serve as a “cold carrier”. Samples were resuspended in buffer (8 M urea, 2 mM disodium EDTA, at pH 7.5), to which and equal volume of sample buffer was added. Radiolabelled samples were separated on 4-13% (PGs) or 4-20% (for xyloside‑GAGs) acrylamide gels and 3% acrylamide stacking gel at 60 V overnight. Gels were fixed then dried and were exposed to an imaging plate (Fujifilm BAS-MS 2040 imaging plate) for approximately 4-5 days. Images were developed on a Cyclone Plus Phosphor Imager (Perkin Elmer).
Bas ms 2040 imaging plate
Manufactured by Fujifilm
Sourced in Japan
The BAS-MS 2040 is a high-resolution imaging plate from Fujifilm. It is used for the detection and analysis of radioactive samples in laboratory settings.
Automatically generated - may contain errors
4 protocols using bas ms 2040 imaging plate
1
Proteoglycan Isolation and Visualization
2
Proteoglycan Separation and Visualization
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Proteoglycans labelled with [35S]-sulfate were prepared for SDS-PAGE by isolation through the DEAE-sephacel anionic exchange mini columns. Samples were added to pre-equilibrated columns and then washed extensively with low salt buffer (8 M urea, 0.25 M NaCl, 2 mM disodium EDTA, 0.5% Triton X-100). Proteoglycans were eluted using high salt buffer (8 M urea, 3 M NaCl, 0.02 M EDTA, Triton-X).
Equal counts of proteoglycans (20,000 - 50,000 cpm) were precipitated by ethanol solution (1.3% potassium acetate in 95% ethanol) chondroitin sulfate was added as a cold carrier. Samples were suspended in 20 μl of buffer (8 M urea, 2 mM disodium EDTA, at pH 7.5) and 20 μl sample buffer (0.5 M Tris–HCl pH 6.8, 10% SDS, 50% glycerol, 2-mercaptoethanol, and 0.1% bromophenol blue). Proteoglycans were separated on gradient separating gel [32 (link)] with 4-13% acrylamide separating gels and 3% acrylamide stacking gels and run overnight at 60 V. Methylated protein molecular weight marker (Rainbow™ [14C]) was used. Processed and dried gels were exposed to an imaging plate (Fujifilm BAS-MS 2040 imaging plate) for approximately 4 days. Images were developed on a phosphoimager (Fujix BAS 1000 image plate scanner) and viewed using imaging software (Fujifilm Multi-Gauge).
Equal counts of proteoglycans (20,000 - 50,000 cpm) were precipitated by ethanol solution (1.3% potassium acetate in 95% ethanol) chondroitin sulfate was added as a cold carrier. Samples were suspended in 20 μl of buffer (8 M urea, 2 mM disodium EDTA, at pH 7.5) and 20 μl sample buffer (0.5 M Tris–HCl pH 6.8, 10% SDS, 50% glycerol, 2-mercaptoethanol, and 0.1% bromophenol blue). Proteoglycans were separated on gradient separating gel [32 (link)] with 4-13% acrylamide separating gels and 3% acrylamide stacking gels and run overnight at 60 V. Methylated protein molecular weight marker (Rainbow™ [14C]) was used. Processed and dried gels were exposed to an imaging plate (Fujifilm BAS-MS 2040 imaging plate) for approximately 4 days. Images were developed on a phosphoimager (Fujix BAS 1000 image plate scanner) and viewed using imaging software (Fujifilm Multi-Gauge).
3
Macroautoradiography of Thymidine Uptake
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We added another group, (G) nontreated control mice, to this evaluation.
On day 28, we diluted 2-14C-thymidine (0.185 MBq) in 0.2 ml of isotonic saline and administrated it at a dose of 0.2 ml to the CRD model control group (D), 25 mg/kg administered group (E), 100 mg/kg administered group (F), and nontreated control group (G). Mice were sacrificed 1 h after the tail vein injection of 2-14C-thymidine and then rapidly frozen in dry ice and acetone. We removed the hind leg surgically from the hip joint using clippers and exposed the femur and tibia. Mice from which the hind legs were removed were embedded in carboxymethylcellulose gel. Twenty micrometer-thick serial sections were made through the sagittal plane of each mouse with the tape-sectioning method using a Cryo Polycut cryostat (Reichert-Jung, Nussloch, Germany) at −20°C. Sections on adhesive tape (Yu-Ki Ban, Nitto Medical Co., Ltd., Osaka, Japan) were desiccated and placed on a BAS-MS2040 imaging plate (Fujifilm Co., Ltd, Tokyo, Japan) for 1 day. Whole-body macroautoradiographs were processed digitally with an FLA7000 image analyzer (Fujifilm Co., Ltd).
On day 28, we diluted 2-14C-thymidine (0.185 MBq) in 0.2 ml of isotonic saline and administrated it at a dose of 0.2 ml to the CRD model control group (D), 25 mg/kg administered group (E), 100 mg/kg administered group (F), and nontreated control group (G). Mice were sacrificed 1 h after the tail vein injection of 2-14C-thymidine and then rapidly frozen in dry ice and acetone. We removed the hind leg surgically from the hip joint using clippers and exposed the femur and tibia. Mice from which the hind legs were removed were embedded in carboxymethylcellulose gel. Twenty micrometer-thick serial sections were made through the sagittal plane of each mouse with the tape-sectioning method using a Cryo Polycut cryostat (Reichert-Jung, Nussloch, Germany) at −20°C. Sections on adhesive tape (Yu-Ki Ban, Nitto Medical Co., Ltd., Osaka, Japan) were desiccated and placed on a BAS-MS2040 imaging plate (Fujifilm Co., Ltd, Tokyo, Japan) for 1 day. Whole-body macroautoradiographs were processed digitally with an FLA7000 image analyzer (Fujifilm Co., Ltd).
4
In-vivo Biodistribution of Radiolabeled Cetuximab
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After the nano-SPECT/CT studies, HCT-116 bearing subcutaneous tumor nude that had been injected 111In-cetuximab after 48 hours. Whole body of nude mice were collected, frozen, and embedded in OCT compound (Tissue Tek, Sakura, Torrance, CA). Frozen sections were placed in contact with a BASMS 2040 imaging plate (Fujifilm, Japan) for thirty days. After complete exposure, the imaging plate was analyzed with an FLA-5100 reader (Fujifilm, Japan) and Multi Gauge V3.0 software (Fujifilm, Japan).
HCT-116/Luc bearing abdominal nude mice whole body of 111In-cetuximab and 111In-DTPA after injected 72 hours were collected, frozen, and embedded in OCT compound after the nano-SPECT/CT studies. The follow steps described as above paragraph.
HCT-116/Luc bearing abdominal nude mice whole body of 111In-cetuximab and 111In-DTPA after injected 72 hours were collected, frozen, and embedded in OCT compound after the nano-SPECT/CT studies. The follow steps described as above paragraph.
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