For the plasma protein binding studies, the anesthetized animals (n = 3 for each tracer) were injected with [18F]F-DPA (27.0 ± 5.1 MBq) or [18F]DPA-714 (29.8 ± 3.4 MBq). After 15 min, the animals were sacrificed and the blood was collected into Li-heparin tubes and centrifuged (3000×g, 10 min). A 10-μl aliquot of the plasma was spotted onto a TLC plate; following this, the remaining plasma was filtered by ultrafiltration using Microsep 30K Omega (Pall Life Sciences, Mexico) with a cut-off of 30 kDa. A 10-μl aliquot of the resulting supernatant was also spotted onto the TLC plate. The plates were exposed onto an imaging plate (Fuji BAS Imaging Plate TR2025, Fuji Photo Film Co., Ltd., Tokyo, Japan) for approximately two half-lives of fluorine-18 (3.5 to 4 h). After the exposure, the imaging plates were scanned using a BAS-5000 reader (Fuji, Japan) with a resolution of 50 μm and the saved images were analyzed using TINA software (version 2.10 g, Raytest, Isotopenmessgeräte, GmbH, Straubenhardt, Germany).
Tina software
Manufactured by Elysia raytest
Sourced in Germany
TINA software is a versatile circuit simulation and design tool developed by Elysia raytest. It provides users with the ability to create, analyze, and optimize electronic circuits through its intuitive graphical interface. The software's core function is to simulate the behavior of electrical circuits, allowing users to explore and validate circuit designs before implementation.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Prism 6, by GraphPad (3 mentions)
Bas 5000 reader, by Fujifilm (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Ecl method, by Merck Group (2 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Merck Group (1 mentions)
Anti gapdh sc 32233, by Santa Cruz Biotechnology (1 mentions)
Anti gfp sc 9996, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Rabbit monoclonal anti pparα antibody, by Abcam (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
F254 tlc plate, by Merck Group (1 mentions)
Ab136877, by Abcam (1 mentions)
Ab22554, by Abcam (1 mentions)
Ab119501, by Abcam (1 mentions)
Sp5 microscope, by Leica (1 mentions)
17 protocols using tina software
1
Plasma Protein Binding Assay for Radiotracers
2
Western Blot Analysis of Ionotropic Glutamate Receptors
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Total protein (50μg per lane) was fractionated by electrophoresis on 9% or 12% polyacrylamide gels under denaturing conditions,43 (link),44 (link) transferred onto nitrocellulose membranes, blocked for 2h in phosphate-buffered saline (PBS) containing 5% dried skimmed milk powder, and then probed overnight at 4 °C with primary the antibody, diluted 1:1000. After repeated washings, the membranes were incubated at room temperature for 1h with horseradish peroxidase-conjugated goat anti-rabbit
IgG (Sigma) diluted 1:10,000. Each sample was run 3 times and the control sample was present in all runs and therefore used to normalize protein levels44 (link)). Polyclonal, (Chemicon) affinity purified to the specific subunit- GluN1, GluN2A, GluN2B or GluA2 primary antibodies were used. Specific antibody binding was detected using enhanced chemiluminescence (Amersham) and visualized by exposing an X-ray film to the membrane.44 (link),50 (link),51 (link)
The density of the scanned protein bands was calculated using Tina software (Raytest, Straubenhardt, Germany).
IgG (Sigma) diluted 1:10,000. Each sample was run 3 times and the control sample was present in all runs and therefore used to normalize protein levels44 (link)). Polyclonal, (Chemicon) affinity purified to the specific subunit- GluN1, GluN2A, GluN2B or GluA2 primary antibodies were used. Specific antibody binding was detected using enhanced chemiluminescence (Amersham) and visualized by exposing an X-ray film to the membrane.44 (link),50 (link),51 (link)
The density of the scanned protein bands was calculated using Tina software (Raytest, Straubenhardt, Germany).
3
Immunoblot Analysis of GFP-VP26 Protein
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Cellular proteins were extracted from Vero cells using SDS sample buffer 1× (62.5 mM of Tris-HCl, pH = 6.8; Dithiothreitol (DTT) 1 M; 10% glycerol; 2% SDS; 0.01% Bromophenol Blue), and immunoblot analysis was performed using an equal quantity of proteins. The proteins were revolved on SDS 10% polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (BioRad Life Science Research, Hercules, CA, USA). The membranes were probed overnight at 4 °C with specific antibodies to detect GFP-VP26 protein. Specific proteins were detected with a secondary anti-mouse antibody linked to horseradish peroxidase (HRP). GAPDH was used as a loading control. The chemiluminescence was detected by using Western HRP substrate (Merk, Millipore, Burlington, MA, USA). Immunoblot band intensity was quantified by densitometry analysis using the TINA software (version 2.10, Raytest, Straubenhardt, Germany). Anti-GFP (sc-9996) and Anti-GAPDH (sc-32233) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Goat anti-mouse immunoglobulin G (IgG) antibody-HRP conjugate was purchased from Merk, Millipore.
4
Western Blot Protein Expression Analysis
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The cells were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40) in the presence of complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Samples were resolved by SDS–PAGE and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech AB, Uppsala, Sweden). After blocking in 5% non-fat milk, the membranes were incubated overnight at 4 °C with the appropriate primary antibody and then probed, at room temperature for 1 h, with HRP-tagged secondary antibodies. The chemiluminescent signals were detected by ECL method (Millipore, Burlington, MA, USA). Comparative analysis of the bands was performed by quantitative densitometry and concomitant use of the TINA software (version 2.10, Raytest, Straubenhardt, Germany). The normalization was done per each band based on the density of GAPDH signal per each line.
5
Western Blot Analysis of Protein Targets
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Western blot analysis was performed as described previously [16 (link)]. Briefly, 50μg of each protein sample was separated on 10% SDS-PAGE and transferred onto PVDF membrane. The membrane was incubated overnight with a primary antibody against each target protein (mouse monoclonal anti-apoA5 antibody, Santa Cruz; or rabbit monoclonal anti-PPARα antibody, Abcam; mouse monoclonal anti-LDL receptor antibody, Progen Biotechnik) at 4°C overnight. After incubation with an HRP-conjugated secondary antibody, immunoreactive bands were visualized using the enhanced chemiluminescence detection system. Data were quantified by densitometry after scanning, using the TINA software (Raytest, Germany). The results of the target protein were presented relative to GAPDH expression.
6
Western Blot Analysis of Autophagy and Apoptosis Markers
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Cell pellets were collected at the indicated time after infection or transfection, washed in 1X phosphate-buffered saline (PBS) and lysed with cell lysis buffer (Cell Signaling Technology). To detect LC3 protein, the following lysis buffer was used: 65 mM Tris HCl pH 6.8, 4% SDS, 1.5% β-mercaptoethanol. Gels containing different percentages of SDS-polyacrylamide were used: 15% to resolve LC3 forms I and II, 12.5% for caspase-8 and 10% for Atg3 and viral proteins. An equal amount of protein extracts was subjected to Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in polyacrylamide gels, transferred to nitrocellulose membranes (Bio-Rad Life Science Research, Hercules, CA), blocked and reacted with primary antibody and appropriate secondary antibody, followed by chemiluminescent detection. Quantitative densitometry analysis of immunoblot band intensities was performed by using the TINA software (version 2.10, Raytest, Straubenhardt, Germany).
7
Cytotoxicity and Efficacy Evaluation
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The data analysis and the graphical representations were performed using the GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Student’s t-test was used for the statistical analysis of the data presented as means of three independent experiments ± standard deviations (SD). The asterisks (*, **, and ***) indicate the significance of p-values less than 0.05, 0.01, and 0.001, respectively. The 50% cytotoxic concentration (CC50) and half maximal effective concentration (EC50) were calculated from concentration-effect curves by using non-linear regression analysis. Quantitative densitometry analysis of immunoblot band intensity was performed with TINA software (version 2.10, Raytest, Straubenhardt, Germany).
8
Western Blot Quantification Protocol
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Protein lysates were collected in RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40) supplemented with complete protease inhibitor cocktail (Roche). Samples were resolved by SDS–PAGE and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech AB). The membranes were blocked in 5% non-fat milk and incubated overnight at 4 °C with the appropriate primary antibody. The membranes were then probed with HRP-tagged secondary antibodies at room temperature for 1 h. Immunoreactive proteins were visualized by means of the ECL method (Millipore). Comparative analysis of the bands was performed by quantitative densitometry and concomitant use of the Tina software (version 2.10, Raytest, Straubenhardt, Germany). The normalization was done per each band based on the density of GAPDH signals per each line.
9
Nonparametric Analysis of Raytest TINA Data
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The data from the Raytest TINA software were analyzed using the nonparametric Mann-Whitney U test. A value of P<0.05 was considered to be statistically significant.
10
Quantification of Viral and Cellular Proteins
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The isolation of nuclear and cytoplasmic fractions was performed as reported previously60 (link). Proteins derived from nuclear and cytoplasmic fractions were quantified by ‘DC Protein Assay’ (Bio-Rad), and equal amounts were used for western blot analysis to evaluate the accumulation of both viral and cellular proteins. Resolved proteins by SDS-PAGE were transferred to nitrocellulose membranes (Biorad). The membranes were incubated overnight at 4 °C with the appropriate primary antibody and successively probed with secondary antibodies. Quantitative densitometry analysis of immunoblot band intensities was performed by using the TINA software (version 2.10, Raytest, Straubenhardt, Germany).
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