However much less is known on the role of local protein synthesis in nervous system pathologies, especially those of the CNS. R. Kandiyoti, K.D. This increases the local viscosity, which is one of the reasons behind the longer decay time of Cybesin (Cytate) in cancerous prostate tissue compared with that in normal prostate tissue. To calculate the total translation foci in the soma or in neurites or in any other desired interval disregarding the bin position, values retrieved from each bin of interest were summed up (Figure 1; workflow A; step 5ii). We then analyzed the distribution of RNA granules, measured as SYTO-stained foci, along Tau-positive neurites. Opin. After three PBS washes, cells were incubated for 1 hr at room temperature with fluorophore-conjugated secondary antibodies: anti-mouse Alexa Fluor 594 (1:200, A-11005, Invitrogen, Thermo Fisher Scientific), anti-rabbit Alexa Fluor 488 (1:200, A-21206, Invitrogen), anti-chicken DyLight 350 (1:200, SA5-10069, Invitrogen), anti-rabbit Alexa Fluor 647 (1:200, A-31573, Invitrogen) and anti-rabbit DyLight 405 (1:200, 611-146-002, Rockland Immunochemicals, Pottstown, PA, United States). e.g. Each pixel represents a point in space. (B) Intensity profiles were measured in the longest puromycin- and III tubulin-positive neurite from randomly selected cells as exemplified. 16-bit images were coverted to 8-bit and binarization was performed using the MaxEntropy mask. RNA localization and localized translation are especially relevant in polarized cells like neurons where neurites extend dozens to hundreds of centimeters away from the soma. We first performed edge detection to find discontinuities in our puromycin labeling that could result from a punctate staining arising from discrete positive foci. The picosecond temporal fluorescence anisotropy I(t) of Cybesin in stained prostate tissue was calculated using Eq.12.7, and the results are displayed in Fig.12.7(b). More recently, a link between intra-dendritic translation, and Tau mislocalization and hyperphosphorylation has been found (Kobayashi et al., 2017; Li and Gotz, 2017). Neurites from raw images (exemplified as heatmaps in Figure 4A) were straighten and divided into 10 m bins. Biol. Waveguides used for optical applications are basically a dielectric structure with high refractive index and transmission surrounded by another medium with low refractive index. 1, 2, and 3, DMSO-treated cells exposed to puromycin for 5, 10, and 30 mins, respectively; 4, 5 and 6, A-treated cells exposed to puromycin for 5, 10, and 30 mins, respectively. As a first step to quantify RNA translation sites in neurites we first detected de novo production of neuritic proteins by puromycilation/SUnSET (Schmidt et al., 2009; Figure 2A). Hello Javdan , Thankyou for guidence. I am actually following the third link you have attached. The lifetime of the fluorophore, and as a consequence its quantum yield, decreases with increasing quencher concentration and an additional term, kq[Q], is added to the denominator of Eqs (3), (7), and (8), to yield. The main source of fluctuation comes from the molecules diffusing in and out of the examined volume. Graphs represent Log2(#puromycin foci +1) vs. distance. However, 2D fluorescence spectra and 3D EEM data require corrections for spectral bias of all optical components. The reflectance of individual coal macerals also increases with coal rank. Made in Amersfoort, the Netherlands The resulting image is smoothen and binarized with the Mask Entropy mask. (1992). PrevNew arrivalExKine Pro Total Protein Extraction Kit for Animal Cultured Cells/Tissues, NextAlkaline phosphatase and Bone Metabolism. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. To calculate the total fluorescent intensity in the soma, in neurites or in other desired interval disregarding the bin position, values retrieved from each bin of interest were summed up (Figure 1; workflow A; step 5i). If it looks great for your control group but selects very few pixels in your other experimental groups, then you will be biasing your analysis, and your MFI might be brighter in one group than another not because you have weaker intensity, but because your analysis couldn't pick it up. The total fluorescent intensity of the puromycin within the desired interval (soma, neurites, etc) disregarding the bin position is shown as (mean puro intensity; step 5i). Additionally, puromycin hotspots were readily visible in distal sites of the neurites, especially in A-treated cells (4, intensity profile and heatmap in Figure 2B). 5). Similarly, despite detecting a significant accumulation of discrete puromycin foci in both control and A-treated cells after 30 min of puromycin exposure compared to shorter pulses, these were significantly higher when A oligomers were added to the cultures (Figure 4F). 14:547. doi: 10.3389/fnins.2020.00547. Because a pump wavelength of 800nm was used, which is very close to the absorption peaks of the two contrast agents (Pu et al., 2008; Pu et al., 2005), there should be very few multiple scattering photons. Make sure you have area integrated intensity and mean grey value selected (the rest can be ignored). Shorter puromycin pulses were tested to decrease the chances of protein diffusion from the actual translation site. Data represent the average value of 1020 sampled cells per condition shown as individual data points, and the mean and median of 6 independent experiments (n = 6). Inertinites show up as light grey or white. Fluorescence intensity for the raw puromycin signal, represented in a colorimetric scale (Figure 2A), was measured along the longest puromycin- and III-positive neurite in randomly selected cells (16, Figure 2A). However, we did not observe changes between DMSO- and A-treated cells possibly due to the slow pace of the translation machinery after a 24-h treatment. For later time points the apoptotic cells take over and the peak is propagated back to lower fluorescence intensities due to the altered replication of NP. Data represent the average value of 510 sampled cells per condition shown as individual data points, and the mean and median of 7 independent experiments (n = 7). Analyze the results: The data can be analyzed using software to measure mean flourescence intensity. 10:445. doi: 10.1038/s41419-019-1636-8, Rangaraju, V., Lauterbach, M., and Schuman, E. M. (2019). (4a), which holds under the condition in which the optical density is less than 0.5. To quantify the puromycin fluorescent intensity as a measure of protein production (Figure 1; workflow A), the longest puromycin- and III tubulin-positive neurite or puromycin- and Tau-positive neurite from randomly selected cells was straightened with the Segmented Line tool in FIJI/ImageJ: Figure 1. The rotation time r and static fluorescence anisotropy r1 for Cytate in cancerous tissue were found to be 1.6 times and ~5 times larger than those for normal prostate tissue, respectively. EBioMedicine 20, 120126. Graphs show the translation sites represented as Log2 (# puromycin foci + 1) vs. distance (step 9). It is important to understand what corrections are being used, the wavelength range over which they are applicable, and how corrections are being applied by automated software routines. Department of Medical Sciences, University of Aveiro, Portugal, Centre for Neuroscience and Cell Biology, University of Coimbra, Portugal. you should identify those pixels You also have the issue that with this method the signal that you are using to delineate "what is a cell" is also the signal you are measuring. "Time is the most precious gift in our possession, for it is the most irrevocable." You can also create a Graphs view in Flow Jo and then add the Mean Intensity parameter. Proc. In this way, only fluorescent biomolecules stimulated by the laser in the focal point are detected. The rotation time r and static fluorescence anisotropy r1 for Cybesin in cancerous tissue were found to be 1.4 times and ~10 times larger than those for normal prostate tissue, respectively. Thus, the combination of RNA and protein staining techniques followed by image processing and binarization, and object-based colocalization can be successfully used to detect sites of local RNA translation in neurons which might be important to unravel the extent of local changes in early stages of AD and other neurological diseases. Whenever stated, a no-primary-antibody negative control was used. Scale bar, 10 m. To overcome this, evanescent field-based sensing systems may be utilized, as was done by Hofmann etal. Acad. Scale bar, 10 m in images. p < 0.05 indicate a significant correlation. The following is the most up-to-date information related to FIJI (ImageJ): Quantification of 2D Images (Measuring Area, Intensity, etc.). doi: 10.15252/embr.201744045, Rutkevich, L. A., and Williams, D. B. p < 0.05 indicate a significant correlation. The longest puromycin-, Tau-positive neurite was selected from randomly sampled cells imaged with identical settings. Copyright 2020 Gamarra, Blanco-Urrejola, Batista, Imaz and Baleriola. J. Neurosci. Box and whisker graph representing the total fluorescent intensity of the calreticulin staining along 130 m of calreticulin- and puromycin-positive neurites. ScienceDirect is a registered trademark of Elsevier B.V. ScienceDirect is a registered trademark of Elsevier B.V. Jiangxi Science and Technology Normal University, Nanchang, China, Lab-on-chip (LOC) devices for point of care (POC) applications, Medical Biosensors for Point of Care (POC) Applications. Nat. A huge variety of latex beads are available commercially, but for fluorescent semiquantitation purposes, a kit containing a minimum of five populations of beads with increasing fluorescence intensities should be used. The light emitted from stained normal tissue undergoes more distance and scattering than that emitted from the stained cancerous tissue; thus the r1 values of the stained cancerous tissue are much larger than those of the stained normal tissue. Neurites from SYTO-positive cells showed significantly higher levels of fluorescence than those not incubated with the dye (Compare dashed line with -SYTO in graph and neurites 1 and 2 in Figure 5A). Understanding dynamic early changes in the local proteome (axonal, dendritic or synaptic) is in our view crucial to understand basic pathological mechanisms underlying AD and likely other neurological diseases. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). ****p < 0.0001 (interaction); two-way ANOVA. The Threshold interface appears. Diffusion of microbeads inside cells cytoplasm or bacterial biofilms have been successfully demonstrated. Control and A-treated cells were fed with 2 M puromycin for 30 min prior to fixation. ###p < 0.001 5 vs. 30 mins puromycin in DMSO-treated cells; ####p < 0.0001 5 vs. 30 mins and 10 vs. 30 mins in A-treated neurons; ***p < 0.001 DMSO vs. A, 30 mins puromycin; two-way ANOVA followed by Tukeys multiple comparison test. Upvote those who contribute to the discussion. Similarly, in the central nervous system (CNS) intra-axonal protein synthesis induced by A142 oligomers, whose accumulation is central to Alzheimers disease (AD), contributes to neurodegeneration (Baleriola et al., 2014; Walker et al., 2018). Results: The percentage of positive area was 3.658%. In our case, we observed similar results in neurites following bath application of hippocampal neurons with A (Figure 2): the effect elicited by A was visible beyond the canonical ER domain and did not affect the cell body (Figures 2C,GJ). 4- As my study include several groups (control vs disease), how can I set the same threshold to all images? Both quantification methods revealed a significantly distinct distribution of translation sites in neurites in A-treated cells compared to controls (Figure 3E). Exogenous stimulus sensed by neurites influence the local translation machinery and mRNAs are released from RNPs complexes. After culturing hippocampal neurons for 9 days and following 24-h treatments with DMSO or A142, neurons were labeled with puromycin. 3. We particularly focused on colocalized objects resulting from 30-min puromycin pulses, which were higher than for shorter puromycin exposures (Figure 5D). The longest puromycin-positive neurite (Figure 2F; left micrograph) of randomly selected cells was straighten and divided into 10 m bins following the workflow Ai (Figure 1). Are you going to be able to detect cells normally? Have these images been acquired with a colorcamera? Obtain a list of the fluorescent intensities of the samples in question. 2- Is measuring mean value the best way to get feedback about the fluorescence intensity (thereby the protein levels) in the region of interest? Click OK after the check is completed, and select No in the dialog box. Synthetic A142 (Bachem, Bubendorf, Switzerland) was dissolved in hexafluoroisopropanol (HFIP, Sigma Aldrich) to 1 mM, aliquoted and dried. The same method was used to measure RNA levels in neurites stained with SYTO. I would recommend that you download FIJI (if you haven't already) here: https://fiji.sc/. It has a lot of pre-downloaded scientific addons and plug WebYou can do this with a single command: Analyze > Measure. Interestingly some authors have linked intra-dendritic translation and Tau mislocalization and hyperphosphorylation (Kobayashi et al., 2017; Li and Gotz, 2017). After 1 day in vitro (1 DIV) the medium was replaced with growth medium (1 B27, 2 mM glutamine, and 50 U.ml1 penicillin-streptomycin in Neurobasal). Finally, press Control (or command)-M to measure the image. Flow cytometry: This method involves using immunofluorescent staining to quantify the number of cells in a certain population. Finally, regardless of the effect of A142, puromycin intensity was significantly reduced in neurites when cells were co-incubated with the translation inhibitor anisomycin (+ anis + puro, Figure 2A; 5 and 6, Figures 2AC). This technique allows the in situ detection of protein synthesis with an anti-puromicyn antibody. Given the high variability, especially in control cells, we did not detect differences between DMSO and A treatments when focusing on distal sites of Tau-positive neurites either. Use the selection tools to measure the area you want to measure. An alternative approach that is closer to absolute quantitation of binding sites for individual mAbs is use of the so-called "antigen-binding capacity" (or ABC) beads. After image acquisition, the longest puromycin- and III-positive neurite (arrowheads in the first panel) was selected with a segmented line, straighten and divided into 10 m bins with the concentric_circles plugin (steps 2 and 3). Under the condition in which the optical density is less than 0.07, fluorescence intensity is proportional to concentration, and thus it is quite convenient to compare several fluorescence spectra with one another. 3 (Zeiss, Oberkochen, Germany) and Hamamatsu EM-CCD ImagEM (Hamamatsu Photonics, Hamamatsu, Japan) digital cameras. In particular, after a nerve injury, mRNAs are locally translated and newly synthesized proteins contribute to axonal regeneration (Terenzio et al., 2018). The longest Tau-positive neurite was selected with a segmented line and straighten, smoothen and binarized with the MaxEntropy mask (MaxEntropy). Altogether these results indicate that in our system puromycin labeling can be used to detect de novo synthesis of neuritic proteins as previously reported in similar experimental setups (Walker et al., 2018; Rangaraju et al., 2019). To extract and compare the time-resolved parameters of Cybesin in cancerous and normal prostate tissues, the total time-resolved fluorescence intensities I(t) of Cybesin in stained prostate tissue were calculated using I(t)=I||(t)+2I (t) from Eq.12.9, and the results are displayed in Fig.12.7(a). Now select Measure from the analyze menu. We then analyzed the distribution pattern of translation foci along neurites. Fluorescence from the bound analyte was guided through the waveguide and finally coupled back out of the grating to be detected by a photodetector. However, a significantly distinct pattern in protein production induced by A oligomers was detected in Tau-positive neurites following a 30-min treatment with puromycin (Figure 4B). Area (ROI ), Min (ROI ), Max (ROI ), Perimeter (ROI ), Median ( ) Results Results-Set Measurements The thin dashed and thin dotted line profiles display the parallel and perpendicular components, respectively. Determining the level of cellular fluorescence from fluorescence microscopy images in ImageJ. The resulting puncta (cyan, Figure 5E) were scored in 10 m bins covering a distance of 150 m from the edge of the cell body. Reference detector and spectral excitation and emission corrections can be made at the time of data collection, and in some instruments this is done automatically by the instrument software. Lasers, on the other hand, have low divergence, can be focused at a point easily, and integrated into a portable system. 12.6. It must be noted that the values used for fluorescence intensity are not standardized between manufacturers, so this is not a quantitative value in the true sense of the word. Flow cytometry can be used to measure cell populations in both healthy and diseased states. Mol. Files 1, 2, and 3 correspond to red, green, and blue respectively. Proc. Threshold the nuclei and add either use the particle Or is this a signal from some fluorescent dye? Only convert current image; Figure 1; workflow B; step 4). Subcellular protein delivery is especially important in signal transduction and cell behavior, and is typically achieved by localization signals within the protein. Histograms C and D show the effect of stimulation with the tumor cell lysate on the same cells. (H) Spearman correlation between non-assisted (wA; 1, DMSO-; 2, A-treated cells) or assisted quantification (wB; 1, DMSO-; 2, A-treated neurons) of translation sites (# puromycin foci) and protein production (mean puro intensity). WebThe latest news about Fiji Imagej Quantification Of 2d Images Measuring Area Intensity Etc. The dashed and dot-dashed line profiles indicate the r(t) for the stained cancerous and normal prostate tissues, respectively. You then need to make sure you're going to capture the right measurements. Dieterich, D. C., Hodas, J. J., Gouzer, G., Shadrin, I. Y., Ngo, J. T., Triller, A., et al. Struct. doi: 10.1073/pnas.59.4.1284, Graber, T. E., Hebert-Seropian, S., Khoutorsky, A., David, A., Yewdell, J. W., Lacaille, J. C., et al. Published data have reported spot quantification procedures without applying convolution filters. 37 Articles, This article is part of the Research Topic, https://www.frontiersin.org/articles/10.3389/fnins.2020.00547/full#supplementary-material, Creative Commons Attribution License (CC BY). (A) Rat hippocampal neurons were grown for 9 DIV and were treated with DMSO or A142 oligomers for 24 h. Cells were incubated with puromycin for 30 mins and processed for III tubulin (gray) and puromycin immunostaining (heatmaps). [10] wherein a CdS thin-film filter and an (In, Ga)N thin-film blue LED was integrated with a disposable PDMS microfluidic device with Si PIN photodetector substrate. The focal point is confocally imaged on a pinhole behind which a single photon detector is placed. WebThis tutorial shows how to measure fluorescence intensity within multiple regions of This subreddit is place to discuss image analysis, software features, to get help, and to share ideas, papers, resources, projects, and expertise. Cell 148, 752764. When comparing the means of two groups taking two variables into account, two-way ANOVA was used. Neurites were divided into 10 m bins and positive puromycin puncta within each bin were visually scored prior to image conversion (1 and 2 in Figure 3D; wA) or were counted with the particle analyzer in binarized images (1 and 2 in Figure 3D; wB). Nevertheless, we sought to determine if in our system what we had reported as neuritic translation sites did in fact colocalize with neuritic RNAs. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Comparison of time-resolved parameters of Cybesin and Cytate in prostate tissues. The most commonly used materials for waveguides is polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), SU8, silicon nitride, etc. (C) Following workflow B, selected neurites were straighten, smoothen and binarized with the MaxEntropy mask (smooth > binary > MaxEntropy). Control conditions with no puromicyn received only fresh growth medium (vehicle). The graph shows the average intensity of puromycin per condition represented as Log2 (mean puro intensity) vs. distance SEM measured in 6 independent experiments (n = 6). Fluorescence intensity was measured in each bin. Alternatively, you can go to Analyze Set Measurements and check off the box next to Limit to Threshold. Then use Image Adjust Threshold to highlight the area you want to analyze, and then Analyze Measure will give you intensity measurements in just your thresholded area. We found no significant correlation between the fluorescent intensity at each neuritic position and the number puromycin foci scored by visual inspection (wA, Figure 4L). The fluorescence lifetime (f) and initial peak intensity (I0) of Cybesin in stained cancerous and normal prostate tissues can be obtained by fitting the temporal profiles of the total emission shown in Fig.12.7(a) using Eq.12.9. Other edge detectors, Laplacian operators distinct to 5 5 matrices or other background subtraction methods can be used depending on the sample requirements and the researchers criteria. Slide the two slider below to change the marked area. In all cases background, contrast and sharpness were adjusted and set the same in control and experimental conditions. Local protein synthesis enables neurites (both dendrites and axons) to change their proteome in an acute manner in order to adapt to fast environmental changes. Figure 2.2. 4. Asymmetrical beta-actin mRNA translation in growth cones mediates attractive turning to netrin-1. 14, 437446. Divide the total number of intensities by 2, and determine which intensity falls on that spot in the ordered list. Culturing Nerve Cells, 2nd Edn. Bldg C17, Optics Valley International Biomedicine Park, Wuhan, China. doi: 10.1038/nn1775, Li, C., and Gotz, J. https://youtu.be/XkGK7T12OiM. Figure 2. Taking advantage of the fact that SYTO-labeled cells were also labeled with puromycin, after binarizing the images corresponding to both stains we applied the AND function in the image calculator which essentially retrieves the colocalization between objects. 1 and 2: no puromycin incubation in DMSO- and A-treated neurons, respectively. In both cases we found a significant high positive correlation (Figure 3H). (1995a). (L) Spearman correlation between non-assisted [wA (DMSO, A)] or assisted quantification [wB (DMSO, A)] of translation sites (# puromycin foci) and protein production (mean puro intensity). By considering that the fluorescence intensity fluctuates around a mean value: the autocorrelation function can be written as: FCS have been extensively used to extract diffusion coefficient of small fluorescent latex bead in solution, showing the influence of the bead radius or solvent properties. ****p < 0.0001; two-way ANOVA followed by Tukeys multiple comparison test. ****p < 0.0001, DMSO vs. A, 30 mins puromycin; two-way ANOVA followed by Tukeys multiple comparison test. Mononuclear cells were incubated overnight in the presence or absence of a tumor cell lysate and labeled with anti-CD3, anti-CD69, anti-CD16, and anti-CD56. Today we're going to talk about Image Js use in immunohistochemistry. The parameters of f, r, r1 and r0 for Cytate were analyzed and obtained using a procedure similar to that described above. Image analysis (software-based): This method involves collecting images of immunofluorescent staining in predetermined regions of interest, and then analyzing them using specific image analysis software. Continue reading here: Calcium Flux Assay Procedure Using Fluo3, Candida Crusher Permanent Yeast Infection Solution. The first utilizes modified amino acids, such azidohomoalanine, that get incorporated into the nascent polypeptide chain. A typical experiment in which one might want to measure relative fluorescence intensity is shown here, where the effect of tumor cells on the expression of CD69 on resting NK cells and T cells from the same donor was investigated. The larger r1 observed in Cybesin (Cytate)-stained cancerous tissue compared with that in normal tissue (as indicated in Table12.2) can be explained by the higher uptake of Cybesin (Cytate) in cancerous prostate tissue. Static quenching is characterized by the formation of non-fluorescent complexes between the quencher and the fluorophore in its ground state. Check the "Dark background box" (because your signal is bright, your background is black), then scroll through the options in that menu and see what looks good; red pixels are above threshold, and what will be counted. N/A, not applicable. Remember that "free help" costs those who help: Aside from Automoderator, those responding to you are real people, giving up some of their time to help you. 3 and 4: 30 min puromycin incubation in DMSO- and A-treated cells, respectively. Powered by Discourse, best viewed with JavaScript enabled, Thresholding and measuring intensity in fluorescence images. Single molecule detection has also been reached. Measure the mean intensity of a background area and subtract this value The interesting features of r(t) curves shown in Fig.12.7(b) are: (1) the values of fluorescence anisotropy of Cybesin in the stained cancerous tissue are always larger than those of the stained normal tissue throughout the decay time; (2) the profile of r(t) for the Cybesin-stained cancerous tissue shows slightly flatter decay in comparison with the normal tissue. All ImageJ users and developers are encouraged to contribute to the ImageJ documentation resources (, Software Packages Built on Top of ImageJ, 2:Frontmost Window and Window Activation, 3:Image Types: Lossy Compression and Metadata, 5:Replacing Red with Magenta in RGB Images, 10:Reducing Memory Requirements When Importing Images, 13:Transferring Selections Between Images, 15:Applying Auto Brightness/Contrast to Entire Stacks, 17:Brightness/Contrast of High Bit--Depth Images, 18:Embedding Color Annotations in Grayscale Images, Customizing the Float Behavior of IJs Main Window, Assigning Keyboard Shortcuts to ImageJ Tools, Cycling Through the Toolbar Using Keyboard Shortcuts. Example Images (what you want to analyze), Reference Images (taken from published papers), Annotated Mock-ups (showing what features you are trying to measure), Screenshots (to help identify issues with tools or features), Good places to upload include: Imgur.com, GitHub.com, & Flickr.com. doi: 10.1016/j.ebiom.2017.05.012, Koenig, E. (1967). Banker, G., and Goslin, K. (1998). Neurosci. Cultures were maintained at 37C in a 5% CO2 humidified incubator. The fluorescence intensity for detection of fluorescein isothiocyanate (FITC)-tagged albumin molecules almost doubled in the case of PDMS lenses as compared to a flat airPDMS interface. The way to measure fluorescence for a concentrated solution is described in the following section. Dendritic morphology of CA1 pyramidal neurones from the rat hippocampus: II. Select the Analyze menu option, then select the Measure menu option. Edinb. It was classically thought that proteins that support dendritic and axonal functions are synthesized in the soma and then transported to the target compartment at peripheral sites of the neuron. The random rotation of Cybesin (Cytate) molecules contributing to fluorescence will result in the decay behavior of r(t). In practice, a parallel laser beam is concentrated through a high aperture microscope lens on a focal point of <0.5m in diameter. Puromycin-positive discrete puncta were analyzed (with the particle analyzer as exemplified in the intensity profiles from straighten neurites. It doesn't appear in any feeds, and anyone with a direct link to it will see a message like this one. doi: 10.1038/s41594-019-0263-5, Jung, H., Gkogkas, C. G., Sonenberg, N., and Holt, C. E. (2014). FIJI/ImageJ > File > Open (do not autoscale) > Segmented Line (Figure 1; workflow A; step 1) > Selection > Straighten (Figure 1; workflow A; step 2). In the second case, you are somehow converting your images into RGB format along the way, which is not desirable for analysis. This is usually followed by the measurement of the vitrinite reflectance, as the accepted indicator of coal rank. Step 3: Mark the stained areaImageAdjustThreshold. As in the case of the raw images, DMSO- and A-treated neurites were selected with a 20- or 40-pixel wide segmented line and straighten. Some fixed neurons were incubated with 50 g/ml DNAse or RNAse (Sigma) for 10 min at room temperature to assess the selectivity of the SYTO labeling. Figure 2.2 shows the relation between reflectance and the carbon contents of coals. 36, 31203138. Following fixation with a PFA/sucrose mix, cells were stained for puromycin and counterstained with an anti-III tubulin antibody to visualize the neuronal cytoskeleton (Figure 2A). Bolton, J. S. (1901). Cell 158, 11591172. Hi there Nil, Well, you shouldn't take the fluorescence of the whole picture (is meaningless), but of your regions of interest (ROIs) which in this EMBO Rep. 19:e45435. (D) The canonical endoplasmic reticulum (ER) was defined by Calreticulin staining (Carl Ab in intensity profiles). Cells were immunostained with an anti-III tubulin antibody to visualize the neuronal cytoskeleton (gray) and with an anti-puromycin antibody to analyze newly synthesized proteins (heatmaps). Locally translated mTOR controls axonal local translation in nerve injury. 5.2). These findings support a model in which retrograde transport of locally produced proteins leads to pathological, transcriptional changes in the neuronal soma.