Mice - Science topic

Hi Balkrishna VASANTRAO Hatwalkar,

I gave up the use of feeders (rat macrophages) to obtain hybridomas (during selection or cloning) after a few years of practice.

I simply coated 100μl of CFS HI in each well, 10 minutes at RT is enough.

By exploiting 5% of the fusion, I obtained several thousand hybridomas.

Respectfully

u can scan all fields with digital slides then we can check every possible zone

If you inject the AAV into the entire cerebellum at P0, expression can typically be observed between P3 and P5. Simply place the pups under a fluorescence microscope to visualize it. However, the visibility also depends on the type of promoter used in your AAV.

Natalia Leciejewska we tried several approaches...but still cells were not attaching properly to the plate: impossibile for us to proceed with transfection. We kept going with continous cell lines, but next here we are trying again. If you get (or I get) any improvement feel free to share: my email is giulio.preta@bchi.vu.lt.

ok, i found some ref.. You can read in chinese, yes? or translate it to english version.

1. 人胰腺类器官培养手册 https://www.yeasen.com/solutiondetail/1000;

2. 胰腺类器官-诺为生物是eBioscience, Miltenyi,STEMCELL, SunJinLab,LGC Lucigen Biosearch授权代理商,是干细胞,免疫学研究的专业化服务平台 http://www.nwbiotec.com/index.php?m=article&a=new_nav_list&newid=254;

Dear Dr. Lucia Fuentes,

You may try the protocol provided below.

You need to first obtain peripheral blood mononuclear cells (PBMCs) as follows.

1. The mouse may be killed and bled by cardiac puncture into a heparinized syringe with a 25-gauge needle.

2. The blood may be diluted by adding an equal volume of 0.9% saline and layered over a NycoPrep 1.077 animal cushion.

3. You may perform centrifugation at 586 × g (no brake) for 15 min. 

4. Mononuclear cells can then be collected from the interface between the plasma and the NycoPrep cushion.

5. These cells may then be resuspended in Tris-buffered ammonium chloride (TBAC)  lysis buffer, which is made by mixing 0.15 M ammonium chloride and 0.17 M Tris at a ratio of 9:1 before adjusting the pH to 7.2 and filter sterilization.

6. The red blood cells may be lysed following 5 min incubation in TBAC buffer for 5 min at room temperature followed by three washes in RPMI-1640.

7. The PBMCs so obtained are then resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum and incubated for 30 min at 4°C with continuous agitation.

(Please note: It has been observed that monocytes spontaneously and rapidly aggregate in vitro at 4°C, which is called the cold-aggregation procedure).

8. Monocytes then spontaneously sediment. You may perform two more successive rounds of cold aggregation.

Regards,

Malcolm Nobre

Liver is a highly metabolic organ hence you should get enough RNA quantity. If you are facing difficulty in getting RNA then follow this suggestions:

1. Since RNA is very unstable, use fresh tissue for RNA isolation on the same day of sacrifice. If your lab facilities does not allow this, then initially store the tissue in RNA stabilizing solution at fridge for 24hr and then in deep freezer.

2. Use new trizol to crush the tissue, do proper homogenization and during homogenization process frequently keep the homogenate on the ice so that temperature maintains.

3. Use fresh chloroform for differential centrifugation.

4. During isopropanol wash, if you are not getting white RNA pellets after centrifugation, repeat the step by adding more isopropanol and allow to chelate at -20 for 15-20 min before centrifugation.

5. Make sure the life of DNase is not expired.

For detailed protocol you may refer my paper: https://doi.org/10.1016/j.phyplu.2024.100679

Hafeezunnisa Mohammed

Seal the entry points and eliminate attractants.

That's great. Rigorous thinking is a respect for science.

Thank you for your response. However, the issue has been somewhat resolved, so I appreciate your suggestions but will have to decline them. Wishing you all the best in your research.

In order to support the development of new asthma treatments, our Gempharmatech platform has independently developed OVA-induced asthma mouse models and HDM-induced asthma mouse models. HDM-induced asthma model is a type 1 hypersensitivity model with Th2 signaling pathway activation, which can simulate the pathogenesis of asthma in real environment with long slow onset cycle.

Please Connect with me and detailed sharing of our model data with you^^

To get a better chance of success, make sure to design primers so the SNP is at least 50 (prefer 100) nucleotides away from the primer site. That way you can avoid the messy start & end of the sequence.

If you have a sequencing core on site, then you could get the data by the next day.

Dear Laurel,

Which monoclonal antibodies you order will depend on several factors such as for example: (i) on your available budget - recombinant ones will probably be much more expensive than those derived by hybridoma technology, (ii) on the amount of antigen needed to immunize animals, if you decide to go that way. Besides, MoAb are usually used as primary antibodies, because they are very specific against epitopes. In contrast, conjugates (secondary antibodies) are usually polyclonal antibodies, which are labeled with different fluorochromes. So you need to check which secondary antibodies work well in your test system. Of course you may also order fluorescently labelled primary antibodies but here we come back to price. There are many companies that produce antibodies, so you should have no problem with this.

Regards

Sorry I do not know how reliable it is but compared with most lab equipment it is pretty cheap Navneet but if cost is an issue then a mains powered bulb and fitment is probably quite cheap or even putting the mouse on a transilluminator but the torches are almost certainly better for the safety of your eyes

Yes, as long as you adjust for their body weight, it should be fine. I inject with hydroxytamoxifen at 6 weeks, but weigh the mice first and adjust accordingly.

Hello Malcolm Nobre ,

I appreciate your thorough answer.

This has been very useful for me.

I have a question regarding the PBS- why is it necessary for it to be plus Ca++ and Mg++?

I thank you in advance for your help.

The benefit of validating neuromuscular studies in mice with a foot-fault assay depends on the study's objectives and the targeted outcomes. While the foot-fault assay is a common method for assessing motor control and balance, compensatory strategies that mice may develop despite injury can lead to false negatives. This can make results misleading and may require additional analyses. However, if the test is supported by other measurement methods (such as EMG), it can provide a more comprehensive assessment and increase accuracy. Thus, while the foot-fault assay alone may limit the reliability of the results, it can be useful when supplemented with additional measurements. Frankly, both in my own studies and while reading others, I have never found motor coordination assessments in mice to provide truly objective data. I believe these assessments should be approached with caution. Additionally, mice have remarkable recovery rates and impressive abilities to maintain balance while walking, for example. Therefore, especially in spinal cord studies, I consider functional outcomes in rats to be rather weak evidence.

Hanieh Tajdozian Hi, Could you find the solution to this problem? I do have the same issue.

Unfortunately, there is nothing you can do. See these discussions on missing citations in Researchgate (a very incomplete list - it is one of the most frequently asked questions here):

Phil Geis

The bright spots you’re seeing are likely contaminants, might be bacteria, fungal spores, or cellular debris.

In this case my suggestion is that:

1. do Gram staining or bacterial fluorescent stain to check if it’s bacterial or else?

2. Change to fresh sterile culture media to rule out contaminated supplies.

3. Assess cell viability just to ensure the issue isn’t due to the cell stress or death?

Dear İremnur Sarıalioğlu

As you know the TUNEL In Situ Apoptosis Kit (HRP-DAB Method) are very sensitive to detect apoptosis in a single cell. Therefore, one has to be very careful while performing the assay and make sure that you adhere company manual protocol to get the desired results.

As said, you got a good apoptosis signal in the first experiment and now its is not coming to your expectations. I am sure that something went wrong either with your protocol, exposure time, washing and HRP-DAB which is light sensitive. Hence, I suggest you to double check and run one small assay by using positive as well as as negative controls samples only and check whether apoptotic signal appears??.

I suggest you to recall whether the kits components were not kept at RT for longer time or if any mixing of components by using same tips may cause serious issue. Other factors like samples over-fixing, concentration of TdT enzyme, prolonged reaction time may also affect apoptotic signal.

Make sure that:

1. The washing is sufficient, otherwise it will

affect the enzyme activity (such as DNase I and TdT

Enzyme) subsequent experimental operations.

2. After washing the slides with PBS, please carefully blot the liquid around

the sample areas with blotting paper.

4. Keep the sample moist during the experiment to prevent the

failure of the experiment caused by dry slides.

5. Avoid repeated freezing and thawing of the Labeling

Solution and TdT enzyme. Stirring by vortex is not

recommended.

Best

Shail

Dear Viet Ha,

The doses for determining LD50 can be any (to a smaller or larger ‘side’ of the estimated LD50, a minimum of 3 doses, the more they are, the more accurately you will determine the LD50. The mean lethal toxic substance doses depending on species, sex of animals, time of year, etc. can differ from author to author by a factor of 2 or even 4.

Check out my articles, especially the early ones, they will be helpful to you.

And also this article in Russian.

There are many methods for determining the average lethal dose:

I think you can find the formulas on the Internet if you want.

The Behrens method I can send you.

Dear Zohreh Estaki , would you mind to add - as an important information - the source / reference / dye specification(s) of your staining sequence ? also guessing about the fixation: "Immersion-" or perfusion fixation of the spinal cord?

Thank you in advance, regards, W.H.M.

Hello Arina Anikina

Yes. C57Blk6 and C57Bl/6 are the same.

C57BL/6 mouse strain is often referred to as "C57 black 6", "B6", "C57" or "black 6". C57BL/6 mice are not written as C57Blk6 mice.

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. The C57BL/6 strain are diverged into two major groups namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide.

Best.

You can try to use the Analyze Particles to build a binary mask based on threshold and size (assuming your dense-core plaques have higher intensities), it should create a binary image only with your dense-core plaques selected and after that you probably want to use the ROI manager to add regions of interest based on that mask to your original image so that you can measure each plaque intensity individually. To analyse the diffuse plaques you will need a mask similar to what you did above, but with a different threshold and maybe a different size range, and the rest of the procedure is similar. You can always subtract masks on imageJ, so creating a binary image that selects all your plaques and subtract the mask for the dense-core can also work fine to have the mask for the diffuse plaques only. Finally, there's a relatively new segmentation plugin called labkit (https://imagej.net/plugins/labkit/ ) maybe it could help you make the process more automated 😊

Here's a summary of strategies to improve RNA yield and purity from mouse gut samples:

1. **Optimizing Homogenization**:

- Use a **bead homogenizer** (if available) or **freeze samples in liquid nitrogen** and grind them with a mortar and pestle for better cell lysis. Alternatively, mince tissues finely with scissors to make them easier to lyse.

2. **Adjust RNA Extraction Protocol**:

- Extend vortexing time with QIAzol to at least 45 minutes, and consider pre-incubating the sample with QIAzol for 5–10 minutes.

- Perform 1–2 freeze-thaw cycles after initial QIAzol treatment for more effective tissue breakdown.

3. **Improve A260/230 Ratios**:

- Add an **extra 80% ethanol wash** step to reduce contaminants.

- Slightly increase chloroform during phase separation (0.25–0.3 mL per 1 mL QIAzol) for purer RNA separation.

4. **Consistency in Processing**:

- Process samples at a uniform time to avoid yield drops due to time variations.

5. **Additional Cleanup for Guanidine Contamination**:

- Increase ethanol during binding or add a post-extraction cleanup (e.g., using a lithium chloride precipitation) to remove salts.

These steps should help improve RNA consistency and purity without extra equipment.

If you have time-to-event data (e.g., time until death), use survival analysis methods such as Kaplan-Meier survival curves. This allows you to account for censored data (mice that were discontinued or survived until the end of the study).

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.

You can artificially inseminate the mice after synchronizaing their estrous cycle to get the birth at same day. But usually if you start the mating protocol in all mice at the same day, the birth will take place within a couple of days. The key is to have enough males and females in separate cages to properly record and monitor the pregnanacy.

I had the same question today. After diving into the literature, I have the following findings.

As you mentioned, mouse FcgRIV is reportedly expressed on monocytes/macrophages and neutrophils, but not NK cells (PMID: 16039578; PMID: 26497511). Checking FcgRIV expression in ImmGen confirms these findings. Therefore, because NK cells are the classical mediators of ADCC, it seems weird to use FcgRIV activation as the proxy for ADCC activity.

The best explanation I can find for Promega's decision is that other cell types, including macrophages, have ADCC activity (PMID: 34204268 and references therein), which could be mediated by FcgRIV. An upside to using FcgRIV is that it has high affinity for IgG2a/c and IgG2b, but no binding to IgG1.

The big picture is that none of these "bioassays" from Promega or other vendors is really a suitable substitute for in vitro (or in vivo) experiments with actual, primary effector immune cells. IMO, these assays are best understood as generic FcR activation assays, rather than indications of specific Ab-driven effector activity (e.g., ADCC, ADCP, etc.).

I would start by diluting a new primer stock in TE (not water). Set up a pcr with positive control using normal amounts of Mg and primer and also tubes with 2x as much Mg and separately 2x as much primer. Before using all ragents thaw eack reagent and them mix by flicking the reagent tube. Sometimes when thawing frozen materials you get water layer on top and concentrated salt/oligo /protein layer at the bottom and you can pipette out water if you take up the upper layer of reagent. Be sure to dilute your primer in TE in case the water has nucleases in it which chews up the primer. run the samples with a dna ladder to check that this is not a gel problem...if you can see the ladder all is well with the gel

Thank you for your suggestion!

This is the picture of how cells look after 48h post-passaging.

Not a recommendation of quality, but you might want to look at those pAbs offered by both Biorbyt (https://www.biorbyt.com/s100a4-antibody-orb6910.html) and antibodies-online (https://www.antibodies-online.com/antibody/703706/anti-S100+Calcium+Binding+Protein+A4+S100A4+AA+15-101+antibody/)

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.

I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?

The ability of primers to extend PCR amplification from the 7th intron to the 18th and 20th exons can be attributed to the placement of the primers flanking a larger genomic region. This allows for amplification across multiple exons and introns, capitalizing on the continuity of the genomic sequence from the primer binding sites. The primers are likely designed to cover a substantial genomic area, which facilitates the amplification of an extended sequence that includes the target exons, despite initial binding in the intronic region. This approach can be common in genomic studies where spanning multiple regions is necessary for comprehensive analysis[1][2].

[1]

Song, X., Liu, Z., & Yu, Z. (2020). EGFR Promotes the Development of Triple Negative Breast Cancer Through JAK/STAT3 Signaling. Cancer Management and Research, 12, 703-717.

[2]

Zou, L., Yu, L., Zhao, X., Liu, J., Lu, H., Liu, G., & Guo, W. (2020). MiR-375 Mediates Chondrocyte Metabolism and Oxidative Stress in Osteoarthritis Mouse Models through the JAK2/STAT3 Signaling Pathway. Cells Tissues Organs, 208, 13-24.

Please take a look at clone H1.2F3.

PE anti-mouse CD69 Antibody

There are a lot of unknowns here... but specifically 2years in PFA the brains are well fixed, and antigens are most definitely cross-linked, which will require a minimum of a quality antigen retrieval step. The other question should be addressed prior to starting; were these animals just decapitated and immersion fixed in PFA, or did the previous group perfuse them with a saline/PBS flush followed by PFA? IF they didn't do this it is probably not worth the following efforts because anything you get will be compromised by the presents of blood in the tissues. IF they did the perfusions removing all the blood, you may want to try to carefully extract the brains from the calavera and begin a cryo-protection protocol (sucrose and eventually a Glycerin/Glycol solution). This is a critical step; you can't just freeze the extracted brains without cryoprotection. After which you will be able to section for frozen IHC. At this point you still have questions... depending on your target antigens you are severely limited due to their age in PFA. In other words, there are many pitfalls with this attempt and your outcomes will vary greatly depending on a number of unknown variables. Good luck.

Wolfgang,

Thank you so much for your very helpful reply to my problem!! I really appreciate the time you took to help me, which what you said, did help very much!! I had told the PI to bisect the pup then put into fixative, which would greatly help the fixation process. I also told him, if he had any more in the future, I would process them for a longer program.

Again, thank you for your time!!!

Take care,

Cynthia

Govindarajan,

Thank you for your reply! I will suggest to the PI to use Bouin's for the fixative. Your processor solutions are what I use on my Automatic Tissue Processor, VIP5, I was really wanting times for each of those solutions, but thank you for reply.

Take care,

Cynthia

Yue Young,

I've been looking into this same question, now 3 years later, and still don't see that any attempt at an auto-scored grid walking task has been made. The tapered beam task is another behavioral task commonly used in my lab, and a cheap, automated version was created in 2017 (PMID: 28860079). I'd be interested to see something similar made for the grid walking task. Especially for my lab, as these two tasks are primary ones that have been used reliably for a lot of our studies, so automating these would save a lot of time.

It seems like it should be relatively easy to use a motion detector that records every time a fault is made and the foot passes through the grid. Then you'd just need the software or code set up to receive that input and score it automatically.

Have you made any progress in looking into this or finding potential solutions?

Dear Sung-Jun Lee,

Great answer. Thank you very much,

Michael

Why do you think your microbe will "rupture"?

Suggest you validate.

Selection of the diet is critical her So we used Normal chaw diet mixed with higher percentage of Lard or saturated Fatty acids from different sources and feeding may continued for at least 16 or 20 weeks

Hello Adamska Mr

To optimize your staining, both the primary and secondary antibodies should be titrated to ensure minimum background with a maximal specific signal. Fluorescently labeled secondary antibody only control should be used when indirect staining is performed. This control is important for determining whether there is any non-specific binding from the secondary antibody.

To make sense of your data and to draw accurate conclusions, it is important to include proper controls. The below attached link will provide you with more information on flow cytometry controls.

Best.

Hello Ulfuara Shefa

The low 260/230 ratio is because of guanidine salts carry over to the sample. Increased absorbance at 230nm in RNA samples is due to contamination by guanidine thiocyanate, a salt which absorbs very strongly at 220–230nm at moderate or low salt concentrations, and can be present in the lysis buffer or extraction reagent (e.g., TRIzol) used in most RNA purification procedures.

The best approach would be to give more washes to the RNA sample. If you have used TRIzol, try washing with ethanol to desalt it. For silica preps, a few extra washes with 70-80% ethanol should clear the column of salts.

Also, I have listed two methods for desalting RNA.

1. You may use an ethanol precipitation with ammonium acetate, which is used in most cases. The purified RNA sample can undergo two precipitations. To form the pellet, one volume (V) of RNA to 3 V of chilled (–20°C) EtOH and 0.1 V of 4M ammonium acetate are mixed in a 1.7 mL microcentrifuge tube. The sample is placed on dry ice for 30–40 min, and then centrifuged for 30min at 13,000rpm. The supernatant is carefully removed from the RNA pellet, and the pellet is redissolved in RNase-free ddH2O (about 20μl), and the process is repeated. After the supernatant is removed the second time, 500μl of cold EtOH (–20°C) is used to wash the pellet, which is then dried in a speed vac and stored at –20°C until use.

OR

2. Desalting can also be accomplished by dialyzing the sample against RNase-free ddH2O. A 200μl RNA sample (with maximum 200μg) is dialyzed against 4 Litre of RNase-free ddH2O for 3 days with a complete change of ddH2O each day. The RNA is lyophilized to dryness, and then redissolved in nuclease-free water.

Best.

Ly6C⁺ CCR2⁺ inflammatory monocytes are typically involved in acute inflammatory responses and can differentiate into macrophages or dendritic cells at the site of inflammation. CCR2 is crucial for their migration from the bone marrow to sites of inflammation.

Ly6C⁺ CCR2⁻ monocytes are thought to play a role in tissue repair and homeostasis. They can also accumulate at sites of inflammation but are more associated with resolving inflammation and promoting tissue repair

I agree genetic relationship is a key issue, but I think there is more important taxonomic detail available today than just the raw genetic similarity. So the Last Common Ancestor of humans and rats (or mice - or rabbits for that matter) was probably a stem member of euarchontoglires, about 90 million years ago. In other words, mice, rabbits and rats are roughly equally related to humans (so choosing between them might be based more on other characteristics such as sociality, cost etc.).

By contrast, say for dogs, the LCA with humans belongs to the boreoeutheria, with an LCA time perhaps 10-20My earlier, so dogs are somewhat less well matched; and in the other direction, the LCA for old world monkeys and humans would be the stem group of the catarrhini, about 35 Mya, and for chimps maybe around 6Mya (making chimps and monkeys by far the most related, but leading to correspondingly greater ethical issues).

By the way, it's worth noting that there are two sources of indefiniteness in the dates. One is lack of knowledge, heavily affecting the older dates (maybe 10My or so), subject to improvement as more fossils are discovered and better dating is applied. But for the more recent dates, the greater difficulty is that taxonomies on the fine scale are actually networks rather than trees, so that even the definition of LCA is a bit fuzzy. For chimps, for example, a reasonable value could be anywhere between about 4Mya (last exchange of genetic information) and 7Mya (when the trees fully merge). This source of indefiniteness is unlikely to disappear.

here they are, outlined in blue

Mice are preferred in experiments because they are genetically similar to humans, have well-established research protocols, and are easier to breed and house compared to toads.

Animal tissues can be stored at \(-20^\circ\)C for up to 3 months with minimal degradation. For longer storage, up to 6 months, some degradation may occur, and beyond that, \(-80^\circ\)C or lower is recommended to preserve tissue integrity.

Even though autoimmune thyroiditis in mice is often employed as an animal model for human autoimmune thyroid diseases, particularly Hashimoto’s diseases, various experiments induce the condition. Some of the established modeling approaches are as follows:

Spontaneous Mod@@@els:

NOD (Non-Obese Diabetic) Mice: These mice spontaneously develop thyroiditis together with other autoimmune diseases, such as diabetes.

Obese Strain (OS) Chickens: Although these are not mice, OS chickens are a classic model and what has been learnt from this model has been used in mouse experiments.

Immunization with Thyroid Antigens:

Thyroglobulin (Tg) Immunization: Mice are immunized with thyroglobulin preparation associated with an adjuvant such as complete Freund’s adjuvant (CFA). This method elicits a T-cell-type autoimmune response common to Hashimoto’s thyroiditis.

Recombinant TSH Receptor Immunization: This is particularly used for experimental models of Graves’ disease. However, it can also be used in the studies of thyroiditis.

Genetic Models:

Transgenic Mice: Spontaneous thyroiditis can occur as a consequence of targeting excessive expression of cytokines such as IFN-γ to the thyroid gland.

Knockout Models: For instance, spontaneous autoimmune thyroiditis can develop by design knockout of genes responsible for immune tolerance (as CTLA-4).

Adoptive Transfer:

Transfer of T Cells: Transfer of autoreactive T cells from a mouse with induced or spontaneous thyroiditis into a naïve mouse to induce the disease.

Induced Models through Chemical Agents:

Iodine Administration: High iodine intake can exacerbate or induce thyroiditis in genetically susceptible mice, as iodine is known to enhance the autoimmune process in the thyroid.

Environmental Triggers:

Infections or Exposure to Pathogens: Certain infections or pathogen-associated molecular patterns (PAMPs) can be used to trigger or exacerbate autoimmune responses in the thyroid.

Hi Johanna,

You can also get them from Creative Diagnostics (https://www.creative-diagnostics.com/NP-OVAL-256350-219.htm) although they are much more expensive (you get price by online inquiry) than it used to be with Biosearch Technologies. To save money, you could also buy NP-Osu from them and OVA protein from other places separately and conjugate yourself because OVA is much cheaper to get.

Hope this is helpful,

Zhixin

Hello Krzysztof Witek

Yes. The sex of the animal is also important when it comes to producing monoclonal antibodies using mice model. Traditionally, female mice are used for antibody production as these animals can be group housed more successfully than males because females are more docile and less aggressive in social interaction.

Secondly, use young adults for whom the immune response is fairly robust and not affected by previous immune challenges. The robustness of the immune response decreases with age after the period of young adulthood. The recommended age is approximately 6-8 weeks.

Finally, you need to consider the health status of animals used for the production of antibodies. Infectious agents exist that may suppress, modulate, or stimulate the immune system. The use of disease-free animals minimizes the likelihood of cross-reactivity to other antigens the animal's immune system may have encountered.

I have attached below the SOP for the production of monoclonal antibodies in mice.

It will be helpful!

Best.

With atovaquone you're out there on the bleeding edge. Good luck!

thanks

Immature cardiomyocytes can show some sarcomere organization. In a mature cardiomyocyte the sarcomere organization is througout the entire cell. Alpha-actinin antibody is used to visualize the extent of Z-disk formation across the cells. It is hard to tell from a phase contrast image alone whether the cells are fully matur.

https://www.nature.com/articles/s41598-023-31144-3#:~:text=In%20immature%20cardiomyocytes%2C%20sarcomeres%20are,%2DActinin%20(Actn2)%20antibody.

a few ways you can try to get your result: 1) the most of proteins from tissue are matrix and cytoplasmic proteins, you have to remove these proteins to enrich your membrane proteins. To do this, use H2O plus protein inhibitor to homogenize your sample, spin down to remove the soluble proteins and keep the pellet (membrane and nuclear proteins), add RIPA to dissolve pellet (it is better you do homogenization again). 2) try buy solution such as Bio-rad casein for wetern blot to increase the sensitivity of detection.

Yes, the AlphaFold2-predicted structure can be used as a template for model prediction in other software like Swiss-Modeler. While AlphaFold2 generally provides highly accurate predictions, it's not uncommon for models to fail certain validation tests like Verify3D, especially if there are local inaccuracies or regions with low confidence.

If the human protein model from AlphaFold2 is not completely passing Save Server checks, but the mouse AlphaFold2 model does pass, you can use the mouse model as a template in Swiss-Modeler to generate a new human protein model. Swiss-Modeler is a homology modeling tool that uses a template structure to model the target protein.

Here’s how you could approach it:

Using a validated mouse model as a template in Swiss-Modeler might help in overcoming issues faced by the human model generated directly by AlphaFold2.

thank you so much for the feedback :)

Layla Shojaie were you able to solve the problem, i am currently experiencing the same problem

Tessa M Alofs Thank you so much!

Hi,

Those dark spots look like deposits of oxidized hematoxylin that randomly deposited in tissue which happens with old hematoxylin solutions.

I would try filtering the hematoxylin solution before use, and if it is over a year old, I'd replace it.

Good luck!

Hello Hyun Jeong Yoon

You may get yellow colored RNA pellet due to the following.

1. If the sample is high in fat content. As a solution, you may centrifuge the sample before chloroform addition and remove the fat layer on the top.

2. For sample with high amounts of blood, some iron or hemoglobin may remain in aqueous phase giving a yellowish color. As a solution, the blood may be prewashed with ice cold sterile PBS before adding TRIzol .

3. Aqueous phase turns yellow upon addition of isopropanol. As a solution, try a fresh bottle of isopropanol.

4. The use of guanidine thiocyanate that has been exposed to light or unfavorable temperature conditions. Guanidine thiocyanate, particularly in the form of aqueous solution, is extremely sensitive to light and temperature. Undesirable redox reactions occur especially in the weakly acidic medium (pH 4.5 to 7.0), whereby elemental sulfur is formed as a decomposition product. This decomposition reaction, which is noticeable gives an intense yellow color. As a solution, store the reagent at temperatures between 2-8°C. At room temperature, the stability of the reagent is significantly reduced. Avoid any prolonged exposure to light.

Hope this helps!

Best.

Dear Colleague,

I hope this message finds you well. Understanding the pharmacokinetic (PK) differences between rats and mice is essential for interpreting preclinical data and translating findings to human applications. Several factors contribute to these differences, which I detail below:

In conclusion, multiple factors, including metabolic rate, enzyme expression, absorption, distribution, excretion, physiological and anatomical differences, genetic factors, and experimental conditions, contribute to the pharmacokinetic differences observed between rats and mice. Careful consideration and control of these factors are essential for accurate interpretation and comparison of PK data across species.

Should you have any further questions or require additional assistance, please feel free to reach out.

What factors may contribute to the PK differences in rats and mice?