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: The primary antibody used was anti-proNGF (Alomone) and the secondary antibody was goat anti-rabbit HRP conjugated (Jackson laboratories).

Techniques: Transgenic Assay, Mass Spectrometry, Recombinant

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: The primary antibody used was anti-proNGF (Alomone) and the secondary antibody was goat anti-rabbit HRP conjugated (Jackson laboratories).

Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA

Journal: Molecular Cancer Therapeutics

Article Title: Nerve growth factor links oral cancer progression, pain, and cachexia

doi: 10.1158/1535-7163.MCT-11-0123

Figure Lengend Snippet: Anti-NGF therapy decreased cancer proliferation in human cancer cells and mouse models. A. Anti-NGF decreased cell proliferation rate of cultured HSC-3 cells at 24, 48, and 72 hours. B. Anti-NGF treatment did not affect tumor size in the mouse paw model. All the tumor groups had significantly larger paw volumes compared to naïve mice (p<.001). C. Anti-NGF treatment greatly reduced tongue tumor size. D. Ki-67 immunointensity was decreased in a representative tongue tumor following anti-NGF treatment. E. Quantification of Ki-67 positive cells in total DAPI positive cells showed significantly less Ki-67 activity following anti-NGF treatment. Horizontal scale bar = 100 μm.*p < .05, **p < .01, ***p < .001; in figure B p < .001 Naïve + anti-NGF vs. all other three treatment; CL, contralateral paw. Two-tailed Student’s t-test or Mann-Whitney U test was utilized in figures A, C, E; two-way ANOVA multiple comparisons was used in figure B.

Article Snippet: Sections were then incubated with rabbit polyclonal antibodies against NGF (1:100; Serotec, Inc., Raleigh, NC) for 2 hours.

Techniques: Cell Culture, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Cancer Therapeutics

Article Title: Nerve growth factor links oral cancer progression, pain, and cachexia

doi: 10.1158/1535-7163.MCT-11-0123

Figure Lengend Snippet: Anti-NGF therapy decreased cancer-induced nociception. A. Anti-NGF treatment greatly attenuated mechanical allodynia in cancer-bearing paws of mice, but had no effect on basal mechanical sensitivity. Two-way repeated-measures ANOVA showed a significant treatment effect (F=511.677, p<.001), and a time × treatment interaction effect (F=1.661, p<.05); the anti-NGF group was significantly different from all other groups (p<.001). B. In the tongue cancer model, two-way repeated-measures ANOVA showed a significant main effect of treatment (F=10.16, p<.001) with a significant treatment × time interaction (F=2.29, p<.05). Mice treated with anti-NGF do not show the escalation in gnaw-time but shown by the control mice starting post-cancer inoculation day 21. C. Immunohistochemistry of a representative trigeminal ganglion from an anti-NGF treated tongue-cancer mouse shows lower expression of TRPV1, TRPA1, and PAR-2 receptors. Scale bar = 100 μm. D. Anti-NGF treatment markedly reduced TRPV1, TRPA1, and PAR-2 immunointensity in trigeminal ganglia of mice with tongue cancer. *p < .05, **p < .01, ***p < .001

Article Snippet: Sections were then incubated with rabbit polyclonal antibodies against NGF (1:100; Serotec, Inc., Raleigh, NC) for 2 hours.

Techniques: Control, Immunohistochemistry, Expressing

Journal: Molecular Cancer Therapeutics

Article Title: Nerve growth factor links oral cancer progression, pain, and cachexia

doi: 10.1158/1535-7163.MCT-11-0123

Figure Lengend Snippet: Anti-NGF differentially modulates body weight in normal and cancer-bearing mice. A. Two-way repeated-measures ANOVA showed a significant main effect of treatment (F=10.16, p <.001) and time (F=7.63, p<.001). The anti-NGF treated group was significantly different from tumor + PBS and naïve + anti-NGF (p<.001), but was not different from tumor + CL-anti-NGF group (p=.09). Vehicle-controlled tumor mice had significant weight loss from their baseline at day 18 (a). In contrast, no weight loss is shown for anti-NGF treated mice. Note that naïve mice treated with anti-NGF had significant weight loss at day 18 (b). At Day 18 and 21, Tumor + anti-NGF group had significantly larger body-mass compared to tumor + PBS or naïve + anti-NGF groups. B. In mice with tongue tumors, two-way repeated-measures ANOVA showed a significant main effect of treatment (F=11.53, p<0.001) with a significant treatment × time interaction effect (F=2.8, p<0.01). Anti-NGF treatment prevented weight loss starting at day 17. *p < .05, **p < .01, ***p < .001, #p < .05 tumor + anti-NGF vs. tumor + PBS, ##p < .01 tumor + anti-NGF vs. tumor + PBS, ++p < .01 tumor + anti-NGF vs. naïve +anti-NGF, +++p < .001 tumor + anti-NGF vs. naïve +anti-NGF.

Article Snippet: Sections were then incubated with rabbit polyclonal antibodies against NGF (1:100; Serotec, Inc., Raleigh, NC) for 2 hours.

Techniques:

Journal: Molecular Cancer Therapeutics

Article Title: Nerve growth factor links oral cancer progression, pain, and cachexia

doi: 10.1158/1535-7163.MCT-11-0123

Figure Lengend Snippet: Anti-NGF in tongue cancer mouse models decreased plasma TNF-α (A) and IL-6 (B), and increased plasma and fat leptin (C & D). *p < .05, **p < .01, ***p < .001 vs. control. Two-tailed Student’s t-test was used for data analysis.

Article Snippet: Sections were then incubated with rabbit polyclonal antibodies against NGF (1:100; Serotec, Inc., Raleigh, NC) for 2 hours.

Techniques: Clinical Proteomics, Control, Two Tailed Test

Journal: Molecular Cancer Therapeutics

Article Title: Nerve growth factor links oral cancer progression, pain, and cachexia

doi: 10.1158/1535-7163.MCT-11-0123

Figure Lengend Snippet: Correlation of cytokines with cancer progression, nociception, and weight change in the tongue cancer model. A, B, C. Plasma IL-6 is positively correlated with tumor size, gnaw-time, and weight loss. D. TNF-α is positively correlated with weight loss. E, F. Plasma and fat leptin are inversely correlated with weight loss. Open circles: control group. Closed circles: anti-NGF treated group. Simple linear regression was used to test correlation.

Article Snippet: Sections were then incubated with rabbit polyclonal antibodies against NGF (1:100; Serotec, Inc., Raleigh, NC) for 2 hours.

Techniques: Clinical Proteomics, Control

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: JOR Spine

Article Title: Age‐related molecular changes in the lumbar dorsal root ganglia of mice: Signs of sensitization, and inflammatory response

doi: 10.1002/jsp2.1124

Figure Lengend Snippet: List of primary and secondary antibodies used for immunofluorescence analysis

Article Snippet: rabbit anti‐NGF , BosterBio , m00341 , 1.00 , 1:100 , same as above.

Techniques: Immunofluorescence, Concentration Assay