Questions related to Embryos
Thanks to "In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart", I have cultured primary epicardial cells, but after continuing to culture and passage, I found that the proliferation rate of the cells was slow. Does the cell have a requirement for growth density, or is it due to other reasons? Has anyone encountered this problem?
During embryonic life the Pre Sertoli cells usually present a very variable shape of nuclei. I want to know weather the mitosis phase of spermatogenesis initiates at the time of puberty, or in the prepubertal period or in the embryonic life itself.
I am planning a point mutation in adult fibroblasts followed by reprogramming into iPSCs. I realised that the proliferation potential of adult fibroblasts is limited. Because of that, they go into senescence before I mutate them and do further reprogramming. In that case, are murine embryonic fibroblasts better than adult fibroblasts?
I am currently trying to make concentrated samples with a hundred frog embryos to make western blots.
Everything is fine during protein extraction. However, when I add the laemmi buffer, I begin to have aggregates that form. Once heated, my samples turn into blue chewing gum impossible to resurface.
Has this ever happened to anyone?
We want to delete some genes and study their effects on developmental angiogenesis. However, intraperitoneal injection of tamoxifen at a dosage of 50 ug/g causes mass non-specific embryonic deaths at E12.5/13.5.
We are exploring possibilities to adapt our stereo microscope due to the high cost of fluorescence stereomicroscopes.
I recently came across this product (https://nightsea.com/products/stereomicroscope-fluorescence-adapter/). However, I've been unable to find any independent reviews or images taken with this particular adaptation that are not provided by the company itself.
In our research, we inject mutations fused to fluorescence proteins such as RFP and GFP in zebrafish embryo. It would greatly assist us in our work to be able to sort embryos based on their expression levels and capture full-body images of larvae under fluorescence. Has anyone attempted to use this adapter and could provide insight on its effectiveness?
Anyone can tell me if it's really worth it?
I have found research units in the States and Australia, but I cannot find such centers in Europe. Maybe you've ordered an MAE test in some European lab?
Could you developmental biologists help me? When injecting cancer cells into the perivitelline space of zebrafish embryo (48 hpf) and growing them 3 days (+34 degrees), what structure/location of the adult fish is that location most resembling for?
I am trying to isolate clean nuclei in mouse embryonic fibroblasts but have been unsuccessful. The purpose is to do nuclei stiffness measures on the sample. I have so far used Sigma kit (link below) but I don't seem to get clean nuclei. Would anyone have tips from similar experience? Thanks!
Hello everyone! I am trying to find a protocol to isolate microglia from mouse embryos. (the mouse model I use produces homozygous lethal pups) All of the protocols I have read are for adult mouse brains. Does anyone know or have a protocol for younger mice?
We work on cattle embryo development we obaserved divisions in most embryos but fail to develop into blastocyst
Very few very are of excellent grade
Other apper to be degrading
And also seen some cellines in culture
I'm a M.S. student at the University of North Carolina at Charlotte studying fiddler crabs and their embryos/larvae. We've been preserving our samples using RNAlater throughout the summer and I'm now extracting them. I've determined that the RNAqueous Micro Kit is the best one for us to use, and I'm using a NanoDrop for quantification.
I did three extractions from the same clutch, each with 50 larvae. My nanodrop concentrations were as follows: 146.7 ng/uL, 154.4 ng/uL, and 126.5 ng/uL. My 260/280 for each was 1.99, 1.96, 1.98, which from my understanding is good. However, my 260/230 were -1.04, -1.74, -1.12.
I have two questions:
1. Are these RNA concentrations high enough to be sent off for RNA sequencing? I'm new to the world of RNA sequencing so any advice is appreciated.
2. What would cause my 260/230 values to be negative? My understanding is that they should be close to 2.
Hello, fellow researchers! I am trying to develop a project to track the growth and development of embryonic and larval killifish (F. grandis and F. heteroclitus) in varying environmental conditions. Confocal or fluorescent microscopy are methods I am thinking of using, but I am unfamiliar with either of these techniques. I am hoping someone with more experience could help give me the pros/cons of both or provide resources to previous research using either technique so I can familiarize myself with what is possible and the methodology. Thank you for any help you can provide!
I have no nearby mouse facility and cannot bring live mice to my research facility. I therefore have to transport either the euthanized pregnant mouse or the mouse embryos (staged E11.5-E14.5) for 1 hour before even starting the dissection. The embryos are used for explant culture of inner organs, so I want to keep the cells alive.
Does anyone have experience with a good method? I'm considering:
1) Transporting the intact (euthanized) pregnant mouse on wet ice
2) Dissecting out the uterus and transporting it in a tube of PBS OR culture medium on wet ice
3) Dissecting out the embryos and transporting them in a tube of PBS OR culture medium on wet ice
I need to validate a series of reference genes (i.e. housekeeping genes) for my qPCR assay and I need to construct a standard curve for each primer pair to check for efficiency. I am using range of snail embryo tissue samples, as well as a snail adult tissue sample.
Due to the limited quantity of RNA from the embryos, I would prefer to use the adult tissue (of high RNA yield) to conduct the standard curves. However, I am planning to use both embryo and adult tissue samples for validating my primers, but it's likely the adult tissue samples will be excluded from the comparison of expression data.
Thus, my question is: would it be acceptable to use cDNA from the adult tissue to examine primer efficiency (e.g. constructing standard curves) as long as the sample is tested (and included in primer validation experiments), but not used in the final analysis?
My project involves RT-qPCR analysis on preimplantation embryos (2-cell). However, I need to store the embryos before conducting the PCR analysis. The storage duration is up to 3 months. Please help me to find the best method to store the embryos. Thank you.
I work on isolation of extracellular vesicles from mouse embryonic fibroblast grown into classical plates but I would like to increase the yield of the isolated extracellular vesicles, si I was wondering if it is possible by trying other cell culturing methods
Hello, all! I'm performing an experiment in which I'm doing a CoIP in the presence or absence of RNase to see if a protein-protein interaction is dependent on RNA. I am trying to identify a protein pair that is known to interact in an RNA-dependent manner, particularly in mouse embryonic stem cells (mESCs). I was thinking of trying to identify some proteins involved in translation initiation or spliceosome assembly/function, but am unfamiliar with the biology of these complexes.
Any and all help is greatly appreciated!
I want to follow the dynamics of expression of a gene over time (different days during embryonic development) in a specific tissue as well as compare gene expression over time to 2 other tissues. Can I compare dCt values of my gene of interest to the geometric mean of the same two reference gene (and not ddCt)? Can you help me find a reference of this type of analysis I can quote?
Hi, I pulled out mouse embryonic stem cells from -80 (where they have been for a week when I froze them down) and plated them on gelatin plates without feeders. The next day I observe these round structures around the colonies. Can anyone identify what they are? We know its not contamination and my ES media contains antibiotics and plasmocin and is freshly made. And these are not degenerating MEFs since I didn't use feeders in my ell culture.
Thanks in advance for your suggestions!
Peace be upon you all,
I want to know which cell in the human body has the most identical genetic sequence to the cells of that person in embryonic life, with the least mutations.
Can it be the neural cells?
Hello everyone, I would like to synchronise mouse embryonic stem cells in either G1 or G2/M phase in order to study the transcription of a set of genes of interest. I'm having some trouble finding a suitable protocol. Any recommendation is appreciated!
I'm currently performing Agrobacterium transformations of immature wheat embryos for a CRISPR-Cas9 project. I do not have any colleagues with experience of working with wheat transformations and I have several questions to ask. Does anyone here have experience with the protocol and is willing to answer several questions about it?
The protocol is:
Ishida Y., Tsunashima M., Hiei Y., Komari T. (2015) Wheat (Triticum aestivum L.) Transformation Using Immature Embryos. In: Wang K. (eds) Agrobacterium Protocols. Methods in Molecular Biology, vol 1223. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1695-5_15
- Why is the embryo axis removed after two days on co-cultivation media while in other species it is removed immediately upon embryo extraction? What would be the effect of removing it before inoculation?
- Why is the temperature of the first incubation different from the following incubations, and what would the effect be of doing all incubations at the same temperature?
- What is the effect of the centrifugation step, and how can I translate a 10 minute centrifugation at 20 000g to one at 17 000g?
- How does one combat agrobacterium overgrowth?
Hi there! I'm performing IHC experiments on PFA-fixed human day 5 embryos (blastocyst stage). After the staining steps, I perform washing in 0.1% Tween-20 in PBS. Although it is usually fine, sometimes embryos tend to attach to the bottom of the dish during washing. The dish we have for washing are not low-adhesion treated (24-well Falcon™ 353047 – Fisher Scientific (10048760)).
I wonder whether I could supplement the washing buffer (0.1% Tween-20 in PBS) with BSA in order to prevent sticking? Any suggestions also about the concentration of BSA to use? Would this change be innocuous for the rest of the protocol or may induce changes that require further optimization? e.g. antibody concentration.
Is it necessary to dechorionate the early stage zebrafish embryos (<10hpf) for immunostaining? If so, what would be the ideal method to dechorionate them? by treating with pronase or manual with forceps?
Hi! I have a problem with the proper positioning of 4 dpf zebrafish (AB strain) for dorsal imaging under a stereoscopic microscope. After the treatment, some of them have yolk edema so it is not possible to place them perfectly straight, even in methylcellulose or other immobilizing media. Of course, they are already anesthetized with tricaine.
In this experiment, I plan to take dozen or more photos (length and angle measurements) therefore I need some wise solution to deal with it efficiently and quickly. Any tips?
I will appreciate any help!
I am working now with an eGFP female mouse and using her embryos, specifically the embryo's brain.
I want to know if there is any marker I can use in immunohistochemistry to label only maternal immune cells in contrast to neuroblast/neurons and glial cells?
Thanks for the attention!
I have attached the data I am using to this post.
I have two variables: alcohol concentration (0%, 1% and 2%), and qualitative grades I've given to embryo samples after doing ISH (1, 2, 3 and 4) with 4 having the highest gene expression.
I'm trying to statistically analyse between the concentrations. I thought that alcohol concentration was categorical and embryo grading was ordinal - is this correct? so then I carried out a Kruskal Wallis test which gave me this result in R:
"Kruskal-Wallis chi-squared = 24.614, df = 3, p-value = 1.859e-05"
the p-value seems very low- almost too low?
I then wanted to do a post hoc / pairwise analysis to analyse between 0%:1%, 0%:2% and 1%:2%. I'm unsure of which post hoc test to use.
any help would be appreciated!!
On pregnancy day 18 we sacrificed one pregnant rat and we isolated the Hipothalami of the embryos (there were approximately 15 pups). All the Hypothalami were collected in one test tube. They were then further processed.
The isolated primary neurons were then seeded on a 6-well plate (3 Mio/well).
What is the N-number in this case?
Is it 1, since all the embryos derived from the same mother?
Or is it 6, since there are 6 wells?
((This refers to a situation in which double-radical emergence!))
Do you think this is a genetic disorder or a unique trait?
Is antibody produced against fowlpox virus vertically transferred via eggs and further inhibited viral propagation on non-SPF cells?
I am trying to stain stage 17 embryos using phalloidin without any success.
I am fixing the embryos in a 1:1 mixture of heptane/4% PFA after dechorionation in 50% bleach. I devitellinize by hand so I don't have to use methanol as it may interfere with the phalloidin staining. For the staining I use 1:500 phalloidin in PBST at room temperature.
So far I didn't have any success. None of the embryos are stained. I tried varying the Triton-X 100 concentration but this didn't seem to make any difference.
I also tried a heat fixation which seemed to improve the staining but destroyed the structures.
Does any one have a working protocol or any idea what I may have to change to get it working?
I have an idea to use a chicken embryo as a model for endothelial disfunction studies. Who has already worked with the vessels of the embryo. It looks so beautifull and easily accessible for the investigation, but I can't find the nethod of the fixation the vessels just from the eggs for the morphological study. Tell me if you have any ideas too
I have 4 independent variables (types of enrichment, types of tanks, mating pairs, strain of zebrafish) (nominal scale) with 1 dependent variable (fecundity of zebrafish/embryos laid) (scale data). what type of statistical test should i use?
We know that somatic embryo can be used as an artificial seeds, but encapsulation include many kind of material. I ask to researcher to give their experience for us.
I'm looking for publicly available Esrrb immunoprecipitation-mass spectrometry (IP-MS) data performed in mouse embryonic stem cells grown in serum condition.
Would appreciate if someone can share with me the info of such publication / datasets
Thank you all
Many pieces of researches have shown that responding explant percentage, embryo number, plant regeneration and also response rapidity increase in TCL explants compared to larger size explant. Now, I ask all researchers to share their experiences and reasons.
Hello, can someone help me? I am just random undergrad interested in oocytes and early embryos but I know almost nothing about their in vitro cultivation. What is the current state of the art of this topics? Where can I find what are the ingredients and what do they affect? Could you recommended me some reviews, articles, brochures, websites, anything about this topics?
Thank you very much!
I am a MS student at the University of North Carolina at Charlotte and I work with ovigerous fiddler crabs and the potential temperature stresses these crabs or their embryos may be exposed to in the field. An experiment has been set up to determine what temperatures these fiddler crabs are exposed to inside of their burrows while they allow incubation of their embryos.
Does anybody know if there is any correlation between the tide and the temperature fluctuations that fiddler crabs experience inside of burrows? especially while they are incubating eggs?
Dear researchers, I need to know your insight/opinion on the most suitable lysis buffer to be used on mouse preimplantation embryo for preparation prior qPCR. In my case, I just need to preserve/isolate RNA only. In addition, how long can the embryo be keep after adding lysis buffer until it will be used for qPCR? Thank you in advance.
How you select and prepare embryonated eggs for diagnosis the following diseases:a ,Turkey chlamydiosis .b _Laryngotracheitis of layers.
We are conducting 21 days reproductive toxicity test using zebrafish. This experiment requires embryo collection daily. We maintain the ZF in 50L aquarium capcity until they reach 16 weeks of age. For the experiment, we transfer the fish pairs from the main aquarium to experimental tanks (5 pairs/4L water/tank). The temperature, feeding, and light conditions are the same as in the main aquarium.
On the first day, ZF lay embryos in all the tanks. However, from day two, ZF stops laying embryos.
Is there any way to restore/maintain the ZF for embryo collection?
Hi all, I am trying to stain the inner cell mass (ICM) of fixed human embryos with TUNEL to detect apoptosis (In Situ Cell Death Detection Kit, Fluorescein from Sigma-Aldrich) for confocal microscopy analysis. However, I only get to label trophectoderm (TE) cells. In my view, it seems that TUNEL cannot penetrate to the core of the embryo, and even after incubating embryos with 0.05 U/ul DNAse (positive control for apoptosis) only all TE cells are labeled but not ICM cells. Has anyone experienced similar problems before?
I perform fixation in 4% PFA/PBS for 20 min at RT, permeabilization in 0.5% Triton-X100/PBS for 30 min at RT, incubation with my primary and secondary antibodies overnight at 4ºC and 1h at RT, respectively; TUNEL incubation for 1h at 37ºC in humid conditions (9:1 TLM/TE) and DAPI staining for 15 min at RT. Several washing steps of 2 min in 0.1% PBST between markers.
Any kind of comment, suggestion or advice would be very much appreciated.
As it is known, for bulls in semen production centers in many countries around the world, to be used in embryo production with the desired health criteria and health conditions for donor cows are almost the same. According to some researchers: “Health in a bull that produces thousands of doses of semen per year.It is understandable that the criteria are very strict. However, just for a donor cow that can produce 20-30 embryos per year.It is not right to ask for the same health criteria” What is your opinion on this argument?
Hi guys! I would like to know if someone could help me to solve a problem the following problem: After the parthenogenetic activation of oocytes, zygotes are degenerating after 72h of activation. I made some changes in the SOF environment, but there was no positive result. Who can help me, I appreciate it!!
Hi all :)
I am trying to perform smFISH followed by immunostaining on drosophila testes but when I check how the staining looks I have no signal from the smFISH probes (while the antibody is fine). I tried to invert the 2 staining and perform IF first and smFISH after. This improved the smFISH but somehow worsen the IF.
In the past I have worked with Drosophila embryos and I never has any issue in performing smFISH followed by IF, could it be a tissue specific problem? Which approach would you suggest to try?
(smFISH and IF alone work both perfectly fine on testes)
Every now and then when I collect eggs, I notice on day two that some will be clumping together, which makes it hard to sort and separate them. When I look at them under the microscope it looks like little fibrous strings holding them together, and they really clump up. Sometimes all of the embryos in the dish will just end up in one pile that I have to pick apart one by one. The strings also glow under fluorescent light. Has anyone else had a similar problem? Does anyone know what this is?
In my opinion, the embryoinic chambers show resemblance to different Orbitolinid genuses:
1. It has protoconch and deuteroconch and subembryonic chambers similar to Praeorbitolina sp. ( Schroeder, 1965), however in Praeorbitolina deuteroconch is not subdivided by partitions and subembryonic chambers positition are at different angles compared to attached image.
2. In Alpillina sp. (Foury, 1968, emended, Arnaud-Vanneau, 1980)
there are 3 subembryonic chambers but in the attached image only 2 are recognizable in my view.
3. In Neoiraqia sp. (Danilova, 1963), the protoconch and deuteroconch ( subdivided ) and periembryonic chambers are similar to the attached image, however shape of the chambers are different.
4. Despite different embryonic chambers compared to Neorbitolinopsis sp. (Schroeder, 1965), (except the divided deuteroconch) the tangential section and shape of chambers in the tangential section in the attached image are very similar to Neorbitolinopsis, though other Orbitolinids might have similar tangential sections.
As it is known, many factors are effective in increasing the pregnancy success rates after embryo transfer to the recipients in cattle. However, can you share your ideas about other applications that will reduce stress and pain during the transfer, apart from epidural anesthesia? For example, how can the implantation chance of the embryo be increased?
I am having trouble processing whole E18.5 mouse embryos and I was wondering if anyone could provide any suggestions on how long I should be fixing the embryos in formalin and what the best processing protocol is? I have tried removing the limbs and making a slit in the back of the embryo to help reagents penetrate and I am currently processing for 2 hrs and 30 min with vacuum but some of the embryos seem like they are not fully processed. I have also tried 2 hrs and 45 min with vacuum but these embryos came out very shrunken and shriveled and the sections did not stain well. Any suggestions would be fantastic!
I'm looking for some help regarding mounting for zebrafish embryos/larvae for live confocal imaging. It seems most protocols in the literature use low MP agarose; however, our lab does not have this. We can make up 3% methylcellulose. I'm struggling to find a sufficient protocol to describe how to conduct live imaging with zebrafish in methylcellulose so that the larvae (from 2-10 dpf) are positioned correctly/remain anesthetized/don't die.
Currently, we have been using silica gel to encase the larvae in water and Tricaine between two coverslips, but it seems that this method does not provide standardized positioning.
Any help/protocol for this would be much appreciated!
I use tamoxifen inducible cre (cre/ERT2) system to knockout a gene at embryonic day (E11.5). For that I am injecting 1mg 4-hydroxytamoxifen intraperitonealy in 100ul corn oil at E8.5 and dissecting the pregnant female at E11.5 as I am doing some hematopoietic studies.
Most of the time I found 50-60 percent embryos are aborted.
I recently started working with the inducible Cre mice and does not have much expreience with tamoxifen. I look for many papers but I do not find much information injecting at E8.5.
Please share your thoughts and suggestions. I will be very grateful.
I want to calculate the E:S ratio of a dormant seed. After 24 h of imbibition, each seed was cut open along the longitudinal axis with a razor blade, and embryo length was measured.
Now I have to measure whole seed length or endosperm length. Please tell.
so, we always had good working primary cortical neurons preps (embryonic) and no issue with culturing them to DIV10-14. However, the animal facility now moved to a different location and instead of being next door it's now a 10min cycle ride away. Interestingly the issue of sudden neuronal cell death at around DIV10 started around the same time. Has anyone experienced something similar before?
We also started adding AraC on DIV4 at 2uM (final conc.) to the cells as we experienced overgrowth of non-neuronal cells but according to the literature this is a well accepted concentration by the neurons. I leave it on until the next media change which happens 3days later.
I use the new B-27™ Plus Neuronal Culture System from ThermoFisher with 50% media change every 4days. Plating (4-5h) is done in DMEM, 10% FBS, P/S.
I'm currently reading a research on CRISPR/Cas9 application on human embryonic cells and really just wanted to know if sample sizes are required when doing research related to such, or if the size is adequate for the study. Sorry if its a dumb question, thanks for entertaining.
Im quite new to R studio and I want to know what statistical test is best to use to compare percentages of time spent at same embryonic stage in 3 species . I know people have said use chi sqaured test but these domt work for percentages.
Thanks for advance for the help
I want to synchronize mouse embryonic stem cells in G2/M phase in order to study transcription of a gene of interest during mitosis and throughout the cell cycle but I'm a having a hard time finding a protocol for this.
I want to study the embryonic development until the blastocyst stage, but the laboratory we are collaborating with for the mice, proposed us to use CD1 mice embryos instead of C57BL/6 for their own technical reasons. Shall I use them or not
Hi all, I have one question regarding zebrafish embryos. After a specific treatment, I collected embryos 3 days post-fertilization. Due to the high mortality rate, I could collect 3 cohorts as being 10 embryos in each cohort. I will extract RNA and then conduct qPCR. In the related articles, I checked, saw that cohorts generally consist of 20-30 embryos but I have 10 for each cohort. Do you think does it affect gene expression patterns, especially significance values? I know that by extracting RNA from 10 embryos, I decrease the biological variability. However, I am not sure about its further outcomes. By the way, I have also 10 embryos in my untreated/control cohorts and convert the same amount of RNA into cDNA, surely. Any thoughts/comments are appreciated.
I have frozen sections of 4%PFA drop-fixed, 30% sucrose dehydrated fetal livers from E14.5 mouse embryos. I sectioned them both at 10 and 5 microns. When I performed the staining on the 10 micron sections the DAPI was extremely dense and therefore I went thinner on the next round. However in both attempts I could not detect any c-Kit signal despite confirming the antibody does work on whole embryo sections. I am using c-Kit eBioscience cat. 14-1171-82
My questions are:
1. Is it just a matter of hit or miss? To my understanding there is not a robust amount present in the fetal liver at any point in time so maybe it is just a matter of staining enough slides to catch some c-kit positive cells?
2. Is there something extra needed to be done as far as the tissue prep? Most protocols in the lit are very vague. The fetal liver is full of blood cells and maybe I should consider perfusing, but then is it possible I also end up flushing out my cells of interest (HSC)?
Thanks in advance!
I've been doing whole mount in-situ hybridization with very small embryos (~E8.5) that are fairly translucent so they're often hard to see with the naked eye. The protocol involves many washing steps so I'm constantly transferring solutions to and from the microcentrifuge tubes that we put the embryos in. Even when I'm being cautious, use a dissecting scope, and use 10µl pipette tips I still end up losing at least one embryo each time I perform the procedure.
Does anyone have methods they use for easier detection of small embryos within microcentrifuge tubes at the bench top? Laser pointers, flash lights, you name it.
We need Fast Green to electroporate plasmid into mice embryos, therefore we simply need it to see some color in the liquid we are electroporating so that electroporation is more efficient.
I find it a little bit expensive and I've found a way cheaper powder version but I have no idea how much "liquid version" can I get from 10 grams:
Does anyone know how should I dissolve this powder to make Fast Green to electroporate mice embryos?
Thank you very much in advance
I have harvested fibroblast cells from R26p-Fucci2 mouse embryos (mouse line as described in this paper: https://dev.biologists.org/content/140/1/237) and cultured them on plastic, hoping to obtain a batch of cells with fluorescence label indicative of their cell cycle state for further analysis. Before I harvested the fibroblasts and grew them up, I checked the endogenous fluorescence signals from the mouse embryos and I could clearly visualize them in the brain region, developing heart, lungs, etc. However, once I cultured them and passaged the fibroblast, the cells didn't seem to express the fluorescence markers anymore. I have also performed genotyping to confirm the presence of reporter genes in the mouse embryos.
The embryos were bred by crossing positive R26p-Fucci2 mice with WT C57 mice, and fluorescence signal were checked after the embryos were harvested from the mother. The embryos with fluorescence signals were processed as follows: First, the head and limbs of the embryos were removed. Then, the internal organs were removed by cutting the embryos open at the abdomen. All the internal organs including the kidneys, intestines, heart, etc were removed. The carcass of the embryos were then transferred to a new plate, and chopped to small pieces. The pieces were rinsed with 1x PBS and put in 1x TrypLE Express solution (approximately 5mL per embryo) in 4 degrees overnight. On the following day, excess TrypLE Express solution were removed where approximately 2 volumes of the solution were left with 1 volume of embryonic tissue. The mixture were incubated at 37 degrees for half and hour and the washed with 1x PBS afterwards. The cells were pelleted and cultured in DMEM (prepared following instruction from the manufacturer). Initially there were few fluorescence signal (around 10 cells out of 200 cells expressing reporters), but after passage, none expressed the reporter. Is there anything I've done wrong, or any procedures I've mistakenly performed that hindered the expression of the reporters?
Thanks for your time and help in advance.
I have been working on the anti-teratogenic activity of a plant extract towards ethanol induced duck embryos however, many studies use the 14 days incubation period and then remove the embryo for morphological analysis. In my case, I want the eggs to hatch and use Tona's degree of malformation scoring table. What would be the best time to inject the teratogen and the anti-teratogen? Is it Day 0 of incubation?
I'm trying to Whole mount in situ hybridization.
In gastrula stage, cells were easily be torn even I didn't treat the pk solution.
And what is the best way to preserve the gastrula embryo cells during in situ hybridization?
I'm trying to whole mount in situ hybridization in fish embryos.
I fixed fish embryos in 4% PFA in PBS for 2days.
And I found some sharp brown spots in embryo. I've never found that spots before.
Can anyone help me regarding this problem?
I performed RT-PCR on embryonic tissues taken from E10.5, and performed whole mount in situ hybridization (WISH) on embryos from the same stage. When comparing the results from these two experiments side by side, i noticed that there are many discrepancies in expression patterns between these two methods. For example, WISH showed no expression of the gene in the heart, but I was able to detect significant bands in the same embryonic tissue by RT-PCR. Can anyone help me explain this difference?
I am thinking to investigate the roles of a specific gene during the embryonic development stages in a crab species. I was just wondering to ask about the possibility of gene knock-down or over-expression before hatching in aquatic animals.
Dose any have some experience regarding the drug/chemical/RNAi delivery into eggs in any aquatic animals?
I am collecting embryos for histological analysis and need to genotype them beforehand. In the past I have worked with older mouse embryos (E8.5 and up), where it is easy to remove the yolk sac and use it for genotyping. However, I now need to start collecting E6.5 embryos. Given that they are so small, I am not sure whether there is enough material to be able to save the epiblast for histology and use other tissue for genotyping. Does anyone have experience with genotyping E6.5 embyros?