Journal: Biophysical Reports

Article Title: Multimodal single-molecule microscopy with continuously controlled spectral resolution

doi: 10.1016/j.bpr.2021.100013

Figure Lengend Snippet: Multicolor single-particle tracking of axonal transport in live neurons. ( A ) Schematic illustration descripting distal axonal uptake of the different neurotrophic factors and their transportation to the proximal axon and cell body through endosomes. The three neurotrophic factors used in this experiment, proBDNF, BDNF, and NGF, were labeled with Qdot565 ( green ), Qdot625 ( red ), and Qdot800 ( magenta ), respectively. ( B ) False-colored overlay of time-averaged localization (RPA = 180°) full FOV time lapse, processed with background removal. In each frame, four consecutive acquisitions with emission filter switching were taken ( white , no filter; cyan , 575/15; yellow , 605/15; and magenta , 809/81) and 405 nm excitation. The FOV shows the signaling activity in the proximal axons near neuronal cell bodies. Orange rectangle marks the axon presented in ( E ). Scale bars, 30 μ m. ( C ) PSF examples of an endosome transporting all three neurotrophic factors through the axon marked in ( B ). Left: three sequential localization (RPA = 180°) acquisitions with emission filters switching (same color code as in ( B )). Middle: single localization (RPA = 180°) acquisition with no emission filter. Right: CoCoS color-detection mode with no emission filter and optimal dispersion (RPA = 172°); color bar on the right illustrates the spectral-dispersion map (nonaccurate). ( D ) Theoretical spectra of the three Qdots with PSF visual representation for illustration purposes (nonaccurate. For a theoretically calculated dispersed PSF, see Fig. S13 ). ( E ) Example time-lapse frames followed by a kymograph of the marked axon in ( B ), showing the retrograde transport of the endosome presented in ( C ). Left: three consecutive acquisitions per frame are shown with RPA = 180°, 405 nm excitation, and 575/15 ( cyan ), 605/15 ( yellow ), and 809/81 ( magenta ) emission filters. A spatiotemporal mismatch between the acquisitions is clearly visible when the transport velocity is high (0, 3.2, and 6.4 s frames). Right: a single acquisition per frame is shown with no emission filter and with RPA = 172°. At the bottom of each time lapse, a kymograph of the full time lapse is presented, showing a halt in transportation at cell death after 30 s of imaging. Horizontal scale bars, 3 μ m; vertical scale bars, 30 s. ( F ) Example of CoCoS PSFs for each of the protein combinations inside the transporting endosomes as detected in the FOV of ( B ). Left: single acquisition with RPA = 172°. Right: false-color overlay of three consecutive acquisitions with RPA = 172° and emission filters switching.

Article Snippet: Meanwhile, human BDNF-biotin (B-250-B; Alomone Labs), human pro-BDNF-biotin (B-256-B; Alomone Labs), and mouse NGF-biotin (N-240-B; Alomone Labs) were mixed separately in a molar ratio of 3:1 with Qdot-625 streptavidin, Qdot-565 streptavidin, and Qdot-800 streptavidin, respectively (Q10131MP; Q10143MP; and Q10173MP; Molecular Probes, Eugene, OR).

Techniques: Single-particle Tracking, Labeling, Activity Assay, Imaging

Journal: Frontiers in Molecular Neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: Recovery of NGF and proNGF spiked in the sample buffer (supplied by each supplier of the commercial kits) .

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: NGF and proNGF spiked into brain cortex extract of proNGF#72 Transgenic Mouse assayed by Emax Promega with mAb Promega and mAb αD11 .

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Transgenic Assay, Concentration Assay, Standard Deviation

Journal: Frontiers in Molecular Neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: IP and WB of transgenic and wild-type mice. (A): IP and WB of cortex extracts from male (M) and female (F) TgProNGF#72 and wild-type (WT) mice. IP on extracts from cortex (CTX) with anti-NGF αD11 antibody, followed by WB with anti-NGF or anti-proNGF antibody, as described in Tiveron et al. . A representative WB probed with anti-proNGF (PAb Alomone), (top) or anti-NGF M20 (Santa Cruz) (bottom) is shown. TgproNGF#72 and wild type mice, male and female, were analyzed. (B) Quantitative analysis of proNGF and mature NGF in the CTX of TgproNGF#72 mice, male and female, by IP and WB and densitometric analysis. After anti-NGF IP, the proNGF bands, (in WB probed with anti-proNGF), and the NGF bands, (in the WB probed with anti-NGF antibody), both identified also by Mass Spectrometry, were quantified. The resulting intensities were normalized against the area of the bands, and then compared with an internal standard of recombinant proNGF and NGF. Loaded samples were in the linear range of detection. Comparison between proNGF and NGF amounts in TgproNGF#72, male and female, is reported in the histogram. The experiment was carried out in triplicate.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Transgenic Assay, Mass Spectrometry, Recombinant, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: NGF and proNGF spiked in the sample buffer (supplied by each supplier of the commercial kits) . The histograms summarize the results of the NGF and proNGF spiked separately and together into the assay buffer of the three different commercial kits for the detection of NGF analyzed. The Promega kit was tested in the two different formats: rat mAb Promega and αD11. Two concentrations of recombinant NGF (10 and 100 pg/ml) and proNGF (50 and 200 pg/ml) were spiked into the assay buffer either alone or together with NGF and measured by using the kits previously described. (A) (Emax Promega with mAb Promega), (B) (Emax Promega with mAb αD11), (C) (Chemikine Chemicon), (D) (NGF Rapid ELISA Biosensis) report the values of concentration, interpolated by the calibration curves. The calculated values of the samples spiked with both NGF and proNGF were compared to the spiked value of only NGF. The t -student test was carried out and the p -values were calculated. Two asterisks on the histograms mean a p < 0.001.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: ELISA for the differential detection of NGF and ProNGF . (A) Strategy: capture NGF and measure proNGF (Exploiting the mAb αD11 fast kinetics). ELISA sandwiches format based on a pre-capturing of NGF by a treatment of the sample with mAb αD11 (Cattaneo et al., ) on a solid support. Subsequent detection of proNGF by traditional sandwich ELISA. Antibodies tested in different combinations: Anti-NGF pAb H20 (Santa Cruz no. sc-548), Anti-proNGF scFV FPro10 (Paoletti et al., ), Anti-NGF pAb M20 (Santa Cruz no. sc-549), Anti-NGF pAb (Sigma no. N 6655), Anti-proNGF pAb (Sigma no. P 5498), mAb αD11 (Cattaneo et al., ), Anti-NGF mAb 256 (R&D no. MAB256). (B) Strategy: capture proNGF and measure proNGF (Exploiting anti—proNGF antibodies in ELISA sandwich). Antibodies tested in different combinations: Anti-proNGF scFV FPro10 (Paoletti et al., ), mAb αD11 (Cattaneo et al., ), Anti-NGF pAb M20 (Santa Cruz no. sc-549), Anti-proNGF Novus (no. S-080-100), Anti-NGF mAb 256 (R&D no. MAB256), Anti-proNGF mAb (clone EP1318Y) (Millipore no. 04-1142), Anti-proNGF pAb Chemicon (Millipore no. AB9040), Anti-proNGF pAb Alomone (no. ANT-005), Anti-NGF Abnova (no. PAB0755).

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA

Journal: Frontiers in Molecular Neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: SPR analysis—calibration curves obtained with proNGF and NGF over the panel of different antibodies. (A) Different proNGF concentrations tested on anti-proNGF mAb FPro10. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (B) Different proNGF concentrations tested on anti-proNGF mAb Millipore. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (C) Different proNGF concentrations tested on anti-NGF mAb αD11. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (D) Different proNGF concentrations tested on anti-NGF mAb R&D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1; (E) Different NGF concentrations tested on anti-NGF mAb R&D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: Binding analysis of NGF and proNGF mixtures to different antibodies . The following antibodies were immobilized on the chip for SPR experiments. (A) anti-proNGF mAb FPro10; (B) anti-proNGF mAb Millipore; (C) anti-NGF mAb αD11. In all the panels, the curves represent the following analytes from top to bottom: 200 nM proNGF + 20 nM NGF (solid blue line, experimental value); 200 nM proNGF (solid yellow line, experimental value); 200 nM proNGF + 20 nM NGF (segmented blue line, theoretical value); 40 nM proNGF + 20 nM NGF (solid purple line, experimental value); 40 nM proNGF (solid orange line, experimental value); 40 nM proNGF + 20 nM NGF (segmented purple line, theoretical value); 20 nM proNGF + 20 nM NGF (solid green line, experimental value); 20 nM proNGF (solid red line, experimental value); 20 nM proNGF + 20 nM NGF (segmented green line, theoretical value); 20 nM NGF (solid dark blue line, experimental value). The segmented lines represents the theoretical curves for the point-to-point algebraic sum of the experimental curves of the single components, at the concentrations indicated.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Binding Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: Calculations of the theoretical value of the measured RU for the single NGF or proNGF components and comparison to the experimental one .

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: AlphaLISA experiments: curves of NGF, proNGF, mix of NGF + proNGF, comparison at different incubation times . Curves of NGF, proNGF, mix of NGF + proNGF, linearly interpolated. NGF and proNGF were assayed separately and together (dynamic range: 2000–4 pg/ml, 1:2 dilutions, in duplicates), in different stoichiometric ratio. The dilution were done in AlphaLISA NaCl buffer (Perkin Elmer). The protocol is described in the methods sections and called for two incubation times. In (A) the first incubation time lasted 60 min and the second one 30 min, as suggested by the manufacturer's protocol. In (B) , a comparison between different incubation times is shown. The first incubation time was 15, 30, or 60 min, while the second one was 20 min for all the curves. The signal was read on the Perkin Elmer instrument EnVision®.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Comparison, Incubation

Journal: Frontiers in Molecular Neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: proNGF CUSABIO kit: Spiking of recombinant mouse proNGF into TgProNGF#72and WT mice brain samples .

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Recombinant, Standard Deviation

Journal: Frontiers in Molecular Neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: Analysis of transgenic and wild-type mice samples by SPR . Injection of samples from transgenic and from WT mice over the FPro10 anti-proNGF antibody (A) and over the Millipore anti-proNGF antibody (B) (blank subtracted curves). Representation of the SPR curves after subtraction of the WT mice signals. Curve of TgproNGF#72 mice subtracted the curve of WT for the FPro10 anti-proNGF antibody ( A —insert), and for the Millipore anti-proNGF antibody ( B —insert). In all panels: Red curve: TgproNGF#72, blue curve: WT.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Transgenic Assay, Injection