Retina - Science topic

Intricacies perfusio with cold fixative for five minute before isolating the retina has given us better results. Try it on a non experimental animal. Best.

Dalam dunia yang didominasi oleh teknologi, layar digital telah menjadi bagian integral dari kehidupan sehari-hari. Dari pekerjaan hingga hiburan, interaksi dengan perangkat elektronik tak terelakkan. Namun, di balik kemudahan ini, muncul ancaman yang tak terlihat tetapi nyata: paparan berlebih terhadap cahaya biru. Kondisi ini telah dikaitkan dengan berbagai masalah kesehatan mata, seperti digital eye strain, degenerasi retina, hingga risiko gangguan penglihatan jangka panjang. Di sinilah peran nutraceutical berbasis alami, seperti daun kelor (Moringa oleifera), menjadi sangat relevan.

Daun kelor, dikenal sebagai "pohon keajaiban," mengandung berbagai senyawa bioaktif yang bermanfaat, seperti beta-karoten, quercetin, dan isothiocyanate. Senyawa-senyawa ini memiliki potensi besar dalam mendukung kesehatan mata, terutama melalui interaksi dengan mikrobiota usus. Penelitian terbaru menunjukkan bahwa mikrobiota memainkan peran kunci dalam mengubah beta-karoten menjadi retinal, komponen utama dalam regenerasi pigmen visual rhodopsin. Artikel ini akan membahas bagaimana sumbu usus-mata bekerja, menjelaskan peran daun kelor sebagai nutraceutical alami, serta mengungkap mekanisme molekuler yang mendasari efek protektifnya terhadap retina.

Mikrobiota: Mediator Utama di Balik Keajaiban Daun Kelor

Peran Mikrobiota dalam Biotransformasi Beta-Karoten

Mikrobiota usus adalah ekosistem kompleks yang terdiri dari triliunan mikroorganisme, termasuk bakteri, virus, dan jamur. Fungsi utama mikrobiota bukan hanya membantu pencernaan, tetapi juga memainkan peran vital dalam metabolisme senyawa bioaktif. Salah satu contohnya adalah biotransformasi beta-karoten, pigmen karotenoid yang ditemukan dalam daun kelor, menjadi retinal melalui enzim beta-karoten-15,15'-dioxygenase.

Retinal, produk metabolisme ini, adalah komponen kunci dalam pembentukan rhodopsin, pigmen visual yang bertanggung jawab untuk penglihatan dalam kondisi cahaya redup. Proses ini menunjukkan bagaimana mikrobiota tidak hanya mendukung kesehatan usus, tetapi juga memberikan dampak sistemik yang signifikan, termasuk pada kesehatan retina.

Metabolit Lainnya: SCFA dan Dampaknya pada Retina

Selain retinal, mikrobiota juga menghasilkan short-chain fatty acids (SCFA) seperti butirat, asetat, dan propionat melalui fermentasi serat daun kelor. SCFA ini memiliki efek anti-inflamasi dan antioksidan yang signifikan. Dengan menekan jalur inflamasi seperti NF-κB dan merangsang aktivasi jalur antioksidan Nrf2, SCFA membantu melindungi retina dari stres oksidatif dan inflamasi kronis.

Regenerasi Rhodopsin dan Siklus Visual

Rhodopsin adalah pigmen visual yang ditemukan di sel batang retina. Pigmen ini memungkinkan kita untuk melihat dalam kondisi cahaya rendah. Namun, rhodopsin tidak dapat berfungsi tanpa retinal, yang dihasilkan dari metabolisme beta-karoten. Ketika rhodopsin terpapar cahaya, ia mengalami perubahan struktur dan memicu serangkaian sinyal saraf yang diterjemahkan otak menjadi gambar. Proses ini membutuhkan regenerasi terus-menerus dari retinal, yang didukung oleh konsumsi beta-karoten dan biotransformasi oleh mikrobiota.

Efek Antioksidan dan Anti-Inflamasi

Senyawa bioaktif dalam daun kelor juga memberikan perlindungan tambahan melalui mekanisme molekuler yang kuat:

Kultur sel retina digunakan untuk mengamati efek langsung senyawa bioaktif daun kelor. Penelitian menunjukkan bahwa ekstrak daun kelor:

Model tikus germ-free dan tikus normal digunakan untuk mengevaluasi peran mikrobiota dalam metabolisme daun kelor. Hasil penelitian menunjukkan bahwa:

Meskipun temuan ini menjanjikan, masih ada tantangan yang perlu diatasi, seperti:

Namun, peluang inovasi tetap terbuka lebar. Dengan memanfaatkan teknologi seperti metabolomik dan nanoteknologi, formulasi berbasis daun kelor dapat dioptimalkan untuk meningkatkan bioavailabilitas dan efektivitasnya.

Penelitian ini menyoroti potensi besar daun kelor sebagai nutraceutical alami untuk mendukung kesehatan mata di era digital. Dengan mengintegrasikan mikrobiota sebagai mediator utama, daun kelor tidak hanya menawarkan perlindungan terhadap stres oksidatif dan inflamasi retina tetapi juga mendukung regenerasi visual melalui biotransformasi beta-karoten menjadi retinal. Pendekatan ini membuka jalan baru dalam pengembangan terapi berbasis mikrobiota yang dapat memberikan dampak positif bagi kesehatan masyarakat.

Dengan pemahaman yang lebih mendalam tentang mekanisme molekuler ini, kita dapat menciptakan solusi berbasis bukti yang tidak hanya efektif tetapi juga ramah lingkungan dan berkelanjutan. Daun kelor, dengan segala potensinya, adalah bukti bahwa alam menyimpan jawaban untuk tantangan kesehatan modern.

According to my Brother a senior surgeon in USA, following is advise:

For lab work like mouse retina dissection, having precise and durable micro-scissors is crucial. F.T.S. 15004-8 is known for its high quality, but if you're looking for alternatives that are more affordable, there are several brands and factors to consider.

Popular Brands:

Dumont: Known for their precision, Dumont scissors are used in various fine dissection tasks. They offer both straight and curved blades, and while they are not the cheapest, their durability and sharpness make them a good investment in the long run.

Sable & Ziegler: Another high-quality brand that offers curved and straight micro-scissors. They are often a bit more affordable than Dumont but still maintain excellent performance for delicate dissections.

Eppendorf: Eppendorf’s micro-scissors are designed specifically for delicate biological dissections and are slightly more affordable. Their quality is often on par with the premium brands like Dumont but can be more accessible for lab budgets.

Vannas: Vannas are very popular in the field for micro-dissections, offering both straight and curved models. They're fairly affordable and long-lasting, often recommended for procedures like yours.

Mandel: Offers cost-effective options that perform well in tasks like retina dissection. They're a great alternative if you're looking for something more affordable without sacrificing too much precision.

Blade Type (Straight vs Curved):

Straight Blades: These are ideal for making clean, precise cuts where you need a flat edge. They're better for cutting through flat tissues or for more controlled dissection.

Curved Blades: Curved scissors allow for more delicate and precise cuts around tight areas or when working on circular or curved structures like retina. Many labs prefer curved blades for tasks like retina dissection because they offer better maneuverability in confined spaces.

Affordability:

While high-end brands like Dumont and Sable & Ziegler can be expensive, there are several affordable alternatives that can still meet your needs for mouse retina dissection. Brands like Eppendorf and Vannas often offer scissors that are priced competitively and still provide excellent quality and precision.

since you want only one as standby or replacement "Bulk Purchasing" options is ruled out.

Please check if suppliers offer discounted price for academic purposes.

For anyone having similar troubles with the ERG procedure, I recommend the following materials:

https://ideaexchange.uakron.edu/cgi/viewcontent.cgi?article=3130&context=honors_research_projects

With the help of Dr. Mark Emerson at the CCNY (http://emersonlabccny.com/), I was able to dissect A. burtoni eye to isolate the retina. The A. burtoni retina is particularly challenging to isolate from the retinal pigment epithelium (RPE) because great care must be taken when peeling away the layer. If mishandled, the RPE can crumble into what looks like grainy particles that are extremely difficult to remove without damaging the underlying retina.

I am still researching whether it is more appropriate to leave the RPE attached to the retina or remove it. On one hand, the RPE increases the signal-to-noise ratio, complicating subsequent analysis. On the other hand, removing the RPE eliminates the retina's recycling station, which impairs cone function.

Additionally, pharmacological agents may be required to isolate specific contributions to the a- and b-waves (as discussed in the papers above), making a fully isolated retina more suitable in such cases.

Overall, I am leaning toward fully isolated retina and hope that conducting an ERG within an hour of retina removal, combined with an appropriate perfusion system, will generate high-quality a- and b-wave data.

This is just a progress update. If anyone has suggestions, please share! Thank you again.

Hi Gudrun,

Thank you for your response. I also suspect that chilling the hot slides immediately lead to tissue detachment or damage, as it happened for me before when I used boiling citrate buffer for retrieval step in IHC.

And when I let slides chill slowly for second run of IHC, there wasn't any detachment.

But the issue is that, the RNAscope protocol says slide transferring to water is immediately. Now, I don't know if I can chill my slide a little before washing or not. I have been waiting for ACD technical support response.

Newer studies have emphasized that hypertension can lead to significant microvascular dysfunction in the retina, characterized by loss of capillary integrity, formation of microaneurysms, and increased retinal vascular leakage. This contributes to retinal edema and may precede visible retinal damage. Recent imaging studies using OCT have revealed more pronounced alterations in specific retinal layers due to hypertension. These include thinning of the ganglion cell layer and increased thickness of the inner nuclear layer, which may correlate with functional visual deficits.

Thanks, Manish Khare, for your question, as well as initiating the discussion board.

As far as one knows, such electric signals you mentioned interact with rods, being afterwards transmitted to the brain, which interprets what is “seen”.

On the other hand, another type of photoreceptors in the retina, called intrinsically-photosensitive retinal ganglion cells (ipRGCs) also perform other functions, such as setting the body’s light-driven circadian rhythms and distinguishing contrast and color in our visual field, which helps processing images on a different level. Further neuroscience studies are yet to discover the origins of such pathways in these cells.

First, represent the Zernike polynomial as a complex polynomial in polar coordinates (r, θ) using the Zernike radial polynomials Rl(p) and angular harmonics Pm(θ). Then, evaluate the polynomial at a grid of points on a circular domain (e.g., using a radial and angular resolution). Finally, use the complex values to create a 2D array representing the surface height at each point. You can use libraries like Python's NumPy and SciPy to perform these steps. For example, you can use the `numpy.meshgrid` function to create a grid of (r, θ) values, and then evaluate the Zernike polynomial using NumPy's `polyval` function.

Based on the relative thickness of the layers, I believe this is right

Key: Outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), retinal ganglion cell layer (RGCL).

Thank you for your suggestion.

Hi Bartosz. Photoreceptor outer segments (OS) are not physically connected to one another in the sense of direct physical connections. Each photoreceptor cell has its own outer segment that is separate from neighboring cells. However, there are specialized structures called interphotoreceptor matrix (IPM) and interphotoreceptor retinoid-binding protein (IRBP) that create a gel-like environment between photoreceptor outer segments, providing structural support and facilitating the exchange of molecules.

In the case of retinal detachment, where the neural retina detaches from the underlying retinal pigment epithelium (RPE), the outer segments can become disorganized and may appear as layers or clumps. This is due to the separation of the neural retina from the RPE, disrupting the normal organization and orientation of the photoreceptor outer segments.

Hi Sonali, here are strategies you can try to improve the purity of your RPE cell population. Here are a few suggestions:

Enzymatic dissociation: Instead of solely relying on mechanical separation, you can try enzymatic dissociation methods to help separate the RPE cells from the retina more effectively. Enzymes like collagenase or trypsin can be used to break down the tissue and facilitate cell dissociation. Optimizing the concentration and duration of enzymatic treatment, as well as the buffer composition, may help improve the efficiency of cell separation.

Cell sorting: If your research facility has access to cell sorting technologies like fluorescence-activated cell sorting (FACS), you can use specific markers to isolate RPE cells directly. RPE cells express markers such as RPE65 or Bestrophin-1, which can be targeted with fluorescently labeled antibodies for sorting. This method can provide highly purified RPE cell populations.

The timing of YFP expression can vary depending on the specific strain of mouse and the type of neuron. In general, YFP expression in Thy1YFP mice can be detected as early as the first few weeks of life. However, the exact timing can depend on many factors, including the specific transgenic line and the individual variability among mice.

Welcome

The way ink mixtures to create a particular hue follows the law of subtractive mixture, as it will reflect what will not be absorbed. (In reality it will reflect, scatter and diffuse what will not be absorbed). The light resulting from the reflection will reach the retina and induce a retinal response, by the means of phototransduction.

You have 2 possibilities:

1. cut off the lense along the Ora serata. If the retina falls out of the eye cup transfer the tissue carefully in another vial containing 4 % PFA or 4% Formaldehyde. Duration of fixation depends of the sice of the retina, mouse rat retinae 3-5 days, bigger once like cat or human 1 week. For cryoprotection put the retina in 30% sucros . Leave it there until the retina lays on the bottom of the vial. Then embed it in OCT.

2. If the retina sticks in the eye cup, fix the eye cup completely with the retina as discribed above. Then peel off the retina out of the eye cup and transfer it into 30% sucrose for cryoprotection.

thank you Dear Marion.

Yes, it would be good if you could isolate the lens, since it could break while sectioning, and breaking the rest of the sections.

Ideally, you would want to use whole- mount retina for everything possible. Especially for quantification/ morphometric analysis. However, some antibodies do not work very well in the flat mount retina. Also, retinal sections being thinner produces better images. In those cases, you would prefer sections.

Using spectral domain OCT, in macular map, assess the ganglion cell complex which includes 3 inner layers of retina namely inner plexiform, ganglion cell and Retinal nerve fibre layer. GCC is damaged in glaucoma and in toxic neuropathy

Thanks Houbin, I've just read your protocol. I've never seen the 1s superglue protocol used before - very interesting. Your sections look beautiful. We use a 2h fix routinely for wholemounts but maybe I should also switch to the shorter fixation time for sections.

Dear all,

Thank you for your answers! It turns out I lost most of my cells on the centrifuge tube wall. My cells landed using the following conditions:

•30k live cells (all cell types), 500µl low adhesion tubes, 3% BSA, swing bucket centrifuge, 200 rcf, 5 min.

•100 cells/µl, viability 75% → 1000 cells/µl, viability 70%

1. Your clinical diagnosis is sufficient for RP and other dystrophies with typical morphology. But you can always get the electrophysiology tests done at centres where they are available. Your nearest medical college should have these facilities. They are also equired for follow up monitoring. But since you are doing these tests infrequently, patient can visit the centers with such facilities at your referral.

But tailoring the management to individual patient depends mainly on the functional vision of the patient and patient's visual needs in terms of his activities of daily life. Use standard questionnaires to assess this. For example this should be helpful

2. LVAs are highly subjective and require specialist assessment. Better start with simple aids like hand magnifier or high plus glasses for reading. Use of smartphones is a good substitute in place of LVAs for most of the patient's visual requirements.

You can try s procedure we have recently developed. Please refer to

and doi: 10.4081/ejh.2020.3154

Hey Salvador,

Its been awhile since I worked with mouse retina RNA extraction and I'm locked out of my old lab protocol I developed. From my memory I used a 250 uL proteinase K digestion for ~10 minutes on frozen tissues, afterwhich I added 750uL of TRIzol LS and further dissociated with bead beating. I followed the standard TRIzol extraction protocol since I found I obtained better total RNA yields. I used an overnight incubation at -80 during the precipitation (which could have just yielded more ribosomal RNA) but I was just doing rt-qPCR. I don't have any published protocol since I left that lab prior to any publications on the approach and no one picked up the project. As I said, this is from my memory of ~6 years ago so you'll definitely have to troubleshoot some. That said, at the time I found that yields by conventional TRIzol extractions yielded better extractions than column based on total recovery, but that could easily be skewed by ribosomal RNA.

Sorry I can't provide you with a more refined protocol to follow!

Dear Rony Ben Zvi Elimelech thank you for sharing this interesting technical question with other RG members. Please have a look at the following potentially useful articles which might help you in your analysis:

and

The first paper is freely available as public full text on RG so that you can download it as pdf file.

I hope this helps. Good luck with your research and best wishes!

I would second what Dimitrolos said. Resin embedding and sectioning is a complex task requiring very specialised skills. Paraffin embedding is a lot more routine and can be done by any histology lab. The choice of method for tissue processing is really down to what you want to do exactly - what stains you want to apply, what you want to evaluate, what is the visualisation technique you will be using, etc... You can see the layer of rods and cones on a standard paraffin-embedded section, but you may not have the level of cellular detail needed for whatever it is you want to evaluate.

In general Bouin's is a better fixative than formalin for eyes, but it's a bit more complicated to use. There is also a modified Davidson's fixative that is commonly used for eyes, you can probably find the approach by doing an online search.

In the cow tapetum lucidum located is located within the choroid layer of the eye, it is found in retina superonasal..

It seems likely that for your purpose, you may just need to add a (freshly diluted) secondary antibody for further incubation without removing an existing secondary antibody (c.f., such action is needed for re-probing with different primary or secondary antibody), but I am wondering whether this helps increase signals (green here). Given your appropriate selection of the secondary antibody (e.g., for species cross-reactivity), an increase in concentration (less dilution) of primary and/or secondary antibody, an incubation time, and an incubation temperature (e.g., from 4˚C to room temperature, or from room temperature to 37˚C) may generally lead to an increase in signals, albeit at the cost of an increase in the background signals (so, the suitable blocking condition is also needed).

So thanks Mr.

Hi,

I m using Axo-clamp 2A for intracellular injection of cortical neurons(S1/V1) with sharp microelectrodes (70Mohms) using 2% biocytin in 1MKCl (even used few times neurobiotin) in bridge mode(its similar to current-clamp mode). I think should be possible with multiclamp 700B too. You need to apply some current pulses to inject dye ( use 2nA current with the duty cycle of 400 msec on and 200 msec off for more than 10 minutes).

I have tried also tried 100Mohms microelectrodes but it was difficult for me to get good labelling with a short injection time. Please note that I m talking about intracellular single-cell injection as per my experience.

Wish you good luck.

Dear Brad Nelson , probably the cheapest solution would be to use a new glass knife. Often the central microscopy facilities at universities or group´s labs have access to a knife maker machine and they also stored the glass strips. Fabrication of new knives from the glass strips is quite easy, you only need to cut the strip in squares and each square in two square triangles. First you need to make a score and then apply pressure on the both sides of the score, so that the glass brokes cleanly through the score line.

Please have a look to the following link where the Leica´s team provided a clear description:

Probably the microscopists at your facilities would be kind enough to help you with this issue.

You also can make a fast search and find some already made glass knives for sale, but I think it would be better to make your own from scratch.

Hope this helps. Good luck with your research work!

In our experiments on frog eyecup preparations the effect of 3 muM tetrodotoxin on the ERG b- and d-waves developed in 10 minutes. Such slow development of the effects was found also for other toxins like picrotoxin (15 min). In the case of tetrodotoxin, an important reason may be the pH dependence of its effects. The time constant of its binding is increased and the efficacy of its action diminished in alkaline pH, because of the reduced availability of its cationic form, which is the active one.

Sorry, but I never heard about it.

Video of intravitreal injection

Vitreous usually comes off very easily in the course of removing the retina. A small piece of Kimwipe can be dragged across the retina, but you must be careful not to also damage the ILM.

What are you trying to do with the cells? As far as I know, there aren't good immortal photoreceptor cell lines. You would need to create a primary culture from retina.

Dear, Brent A Bell Well, I have OCT images of the mouse retina on several instruments including Micron IV, Bioptigen, OcuScience with a few more it can be the foundation for the review. I hope to find few more collaborators to complete the set.

Dear Najiah Musa,

Thanks a lot for your valued answer.

As you know, VNN virus could be a neurotropic virus and can be move to CNS as target tissue. So, it could be logical that it can be localized in the brain.

Then it can move to Retina through Optic nerve and arrive to Eyes and Retina as one of target tissues.

Thanks again

Thank you for your question

But this is not the scope of my work

Greetings to you

@Shubhash Chandra Yadav Vectashield Vibrance and Plus are newer formulations that do not quench Alexa Fluor 647 signals. Only the original Vectashield is not suitable for far-red fluorophores during long-term archiving.

Hi Marc,

Thanks for the information. It is good to know.

Jun

Dear Hendrik,

It has been a while since you asked the question. Did you manage with the experiment?

You could try magnetofection. There are kits available for in vitro and in vivo transfection - you may need a combination of the two. OzzBioscience has a couple of options available. https://www.ozbiosciences.com/8-in-vivo-delivery-transfection. OzzBio also supply si3Dfect transfection reagent which may fit your application. https://www.ozbiosciences.com/3d-transfection/63-si3d-fect-transfection-reagent-silencing-3d-scaffolds.html. I am sure their tech support will be able to help you.

MEA preamplifier stages are usually placed in close proximity to the neuronal cell culture coupled to the MEA substrate to minimize signal attenuation and noise coupling, enhancing the signal-to-noise ratio (SNR) of recordings. This raises the need to limit the amount of produced heat by the circuitry surrounding the array, in order to prevent significant cell culture temperature upward drifts able to perturb neuronal physiology and cell viability (i.e., >38°C) . This issue requires attention mainly in experimental setups integrating climate control capabilities (e.g., portable culturing and recording chamber or cell incubators embedding MEA equipment) to maintain cells viability during prolonged MEA recordings (i.e., >1 hour). Indeed, the encapsulation of the MEA recording equipment in a confined space kept at physiological temperature hinders or slows down thermal dissipation. A common solution to perform climate-controlled recordings is to insert a commercial MEA preamplifier stage (i.e., the MEA1060 device sold by Multi Channel Systems GmbH) inside a cell incubator . However, the power consumption of the MEA1060 (i.e., 2 W) requires the integration of additional devices (e.g., heat sink) in order to neither damage cells due to overheating nor perturb the incubator temperature controller, raising possible issues of sterility and encumbrance. Besides the problem of overheating, the performance of MEA equipment integrated in such setups is downgraded by the high level of humidity (i.e., relative humidity > 90%) traditionally used in cell culture environments to maintain osmolarity and thus cell viability. This imposes to lower the humidity to ambient levels (i.e., <60%) in order not to damage MEA interface boards, which however induces a faster osmolarity increase

This reference might help:

use NRL and NR2E3 antibodies for rod photoreceptor nuclei, Rhodopsin, ROM1, Peripherin2/RDS, ABCA4 (ABCR) for rod photoreceptor cytoplasm.

Use antibodies to CAR (cone arrestin), OPN1sw (short-wave cones, S-cone marker), L/M opsin (low-medium wave cones, - rodents, or separate ab's for L, M cones in primates, humans) -cytoplasmic stain antibodies, RxR gamma -cones, TRbeta2 for L/M cones (rodents), L, M cones -humans. I believe this is the answer you were searching for

I think you mean you have problem with autofluorescence? Do not you use something to eliminate the autofluorescence of blood vessels of any tissue? You can try with Lysine or milk. I do not have a protocol here right now, but it could not be difficult to find it.

Or... Do not you remove the lens during the processing of the tissue before slicing? Sometimes the vitreous comes out with the lens or at least it what happens when I do it in the shark.

Good luck!

Thank you both for the useful information! much appreciated!

Hi Nadim, For the cornea we have several options. You would want to look at the use of 1. Boston Keratoprosthesis 2. Alphacor (https://dx.doi.org/10.1038%2Feye.2011.122) 3. KeraKlear 4. Dohlmans Artifical Cornea. For the retina, you can look at : 1. Argus family of retinal implants 2.Nanoretina NR600

The FDA approval is usually via the Humanitarian Device Exemption (https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfhde/hde.cfm?id=H110002)

Hello Mario, sorry for answering so late.

Our conclusion was that DAPI does not interfere with DAB staining. The main problem is if you store your slides for longer time (2 weeks or more), then it is a big problem to visualize any DAB staining.

So all in all, you can use both together, but take the images as fast as possible.

The default Axial lenght of Topcon DRI OCT Triton is 24.39mm

i agee

Yes, this does look like a legitimate positive signal. I think it's either antibody cross-reactivity with another protein. Check the antibody quality (who purchased, when, what kind of antibody this is, to what region/sequence), if this antibody has been validated and used in your lab for a long time without problems, then it's a legitimate signal and there in fact are GFAP positive cells there in that location.

Raman Nelluri, I've included a retinal photo of the right eye with an explanation of where to measure for the A/V ratio. One disc diameter away from the optic nerve is around the second bifurcation.

Courtney Seligman Please bring your references. By the way, had you read about 'worm holes'?

The staining pattern doesn’t have to be identical between different organs esp. with PI in the mix. The frequencies and subsets of cells expressing PI and your other marker might be different between the different organs. So comparing your plots from heart and retina might not be the best idea.

Do you have a plot from retina that looks different and which you would consider normal? If not, the staining pattern you observe in the retina might be how it is. If you do and this plot looks different and strange, then try to compare the steps in the two experiments and see if there is something different that can explain why you see the strange pattern now. Many times, the odd patterns you see can be corrected by tightening your FSC-SSC gates or by gating out dead cells. That is why I asked to see you initial gating.

I make paraffin sections of whole zebrafish (10 day decalcification in EDTA, 7 day fix in 4% PFA) at 5um and have no problems with detachement or shifting. So it should be possible. If you need my protocol for fixation and sectioning, let me know.

Good luck!

Joke

try adding 0.1% EDTA, and if you don't stain for DNA, maybe use dnaseI. DNA is a sticky molecule. Getting rid of it might improve your readings.

Upload the full source code.

Yes, you can convert a 3D model to a series of cross-sectional images using 3D Slicer. For example, you can load the STL file, then in Data module right-click to convert it to segmentation, then right-click on the created segmentation to convert it to labelmap. Post further questions on using 3D Slicer to discourse.slicer.org.

Glaucomatous field defects such as arcuate scotoma are horizontally oriented correlating with the arrangement of NFs while in retina. The NFs make a 90-degree turn in the ONH and become vertically oriented in the prelaminar region and LC. After the 90 degree turn there is neither horizontal raphe nor are the arcuate NFs. Therefore, the characteristic glaucomatous field defects such as arcuate scotoma and Ronnie's nasal step can't be produced if injury to NFs occurs after their 90 degree turn and become vertically oriented.

Can any researcher please explain how the arcuate scotoma and Ronnie's nasal step be produced if LC is the site of injury - when no such arrangement is present in LC. Thank you in advance for the answer.

Lets assume you have a biometric evaluation system that assigns all authentication attempts a 'score' between closed interval [0, 1]. 0 means no match at all and 1 means a full match. If the threshold is set to 0, then all the users including the genuine (positive) and the impostors (negative) are authenticated. If you threshold is set to 1 then there is a high risk that no one may be authenticated. Therefore, in realtime systems the threshold is kept somewhere between 0 and 1. So, this threshold setting can sometimes may not authenticate the genuine users, which is called FRR (False Reject Rate) but may also authenticate the imposters, which is given by FAR (False Accept Rate).

Here, FP: False positive, FN: False Negative, FN: True Negative and TP: True Positive

FAR is calculated as a fraction of negative scores exceeding your threshold.

FAR = imposter scores exceeding threshold/all imposter scores.

imposter scores exceeding threshold = FP

all imposter scores = FP+TN

FRR is calculated as a fraction of positive scores falling below your threshold.

FRR = genuines scores exceeding threshold/all genuine scores

genuines scores exceeding threshold = FN

all genuine scores = TP+FN

It is going to be tricky without knowing the cell number. You could try to measure the size of your retinal explants and correlate the size to the efficiency. For organoids and neurosphyeres it is done in this approach.

You can process the fluorescence images through the imageJ software. For further details, refer to this link:

Dear Amaranath,

I am sorry, but unfortunately this is not my area of expertise. Maybe the following papers can help you a bit with your question:

Joselevitch C. Human retinal circuitry and physiology. Psychol Neurosci 2008;1(2):141-165. http://www.scielo.br/pdf/pn/v1n2/v1n2a08.pdf

Asari H, Meister M. Divergence of visual channels in the inner retina. Nat Neurosci 2012;15(11):1581-9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717330/pdf/nihms488915.pdf

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Have a good research day, Martin

Jasleen, thank you for your answer. I will take a closer look on the article you recommended. :) Have a nice day!

Hi Reema,

That would be great!

dear Ebrahim,

please insert your question in Google or Google Scholar.

For google :

ca. 520.000 hits.

read the various titles of the hits and perhaps you ´ll find the required analytical tool.

I know it is laborious   but " it is a part of scientific work/ Artur dixit".

JRG

Hi Tim,

Thanks for the question!

Well this cell (and Josh's JAM-B cells also) looks like the one I name to G15 in my characterisation (Volgyi et al. 2009; and it was C6 in Sun et al 2002). I always foun this cell coupled to a cohort of regularly and closely spaced somata in the INL. We concluded that they must have been amacrine cells. I have never seen these cells tracer coupled to either other G15 or displaced amacrine cells though.So, to answer your question I'm not sure what those cell bodies could be in the GCL. However, their shape and size are very similar to your NB injected cell, which is an indication towards homologous coupling. I'll check the literature what kinda coupling patterns have been describend JAM-B cells in the literature and get back to you.

..in case I forget.. please drop me a reminder message!

best,

Béla

I just saw this! i apologize!

in short yes!

Identify the intensity of the control, then subtract that from the intensity of your stained samples

to make it more accurate don't do an overall measurement of your single image. break your image into sections (1cmx1cm) and use them as separate readings for a single image. Then you will be taking account the normal variation you see in any stained section (the intensity of the colour and the depth of the staining varies between different areas of the image)

Use anti-acetylated tubulin. We used it to image neural sprouting in the zebrafish maxillary barbel (see Figure 5):

Since photoreceptor opsins have a rather intense autofluorescence in both red and green channels, one thing I would suggest you is to try using secondary antibodies with far red fluorophores.

Best luck!

Hi Anna,

i might be late but here is some techniques used in current literature and discussion about this topic.

Iba-1 is a Ca channel antigen and CD11b is the "macrophage-1 antigen". when microglial cells detect or injured by reactive oxygen species they started to express CD11b (1st paper) which can be used as a proof of activation. so you can measure the activation by calculating the increase in your fluorescent intensity.

But we always keep in mind that these cells are CNS form of macrophages and they have this CD molecule as proof of their identity which can be found in resting state(2nd paper).

Co-localization of both of these markers (which had been done in 3rd paper) can give you a better clue about activation or you can do cell-quantification by using an image-analyzer software which i believe is to be more accurate as done in 4th paper.

Hello, Steve

Did you end up with using any rabbit anti-MAP to label RGC? Could you share the cat# and the staining quality of the antibodies please?

Thanks!

Dahong

Jax lab is the best way to go, you are on right track