Mice - Science topic

We want to study the effect of High Fat Diet in rats.......

Hi,

I'm working on the immunity of tumor. I injected tumor cells into the mice subcutaneously. After harvesting the tumor, I used percoll and isolated immune cells. Then I added trypan blue and counted cells using a cell counting chamber before flow cytometry staining.

I found in most of the papers, they just said "Count cells for staining". But they didn't say count which kind of cells. Because I saw there were different type of cells in the microscope.

Do I need to count all living cells, no matter how different the shape they are. Or do I just count one specific cell?

Many thanks.

I'm trying to culture hematopoietic stem cells of mice, referring to an article, in which fibronectin-coated plates are used. Since pre-coated plates are expensive, I'm thinking about coating plates by myself.

I found that relatively wide range of amount is on the instruction manuals, such as, concentration of "5-30 ug/ml, 50 ug/ml", amout of "1-5 ug/cm2, 5 ug/m2", or sometimes "depends on the type of the cells". I searched on the internet for the correct answer, but I haven't found one.

Does anyone know the answer?

Hello

I am planning to deliver a cy5-labeled drug to mice, and then check its distribution in different organs. In case I wish to preserve the tissues for later research, will fixation (using 4% formaldehyde) damage the fluorescent signal of the cy5?

Does somebody have any experience with cy5 and fixation?

thanks in advance.

I have isolated cortical neurons from mouse pups ( day1) and seeded on poly d lysine coated wells of 24 well plated (120000cells/well) and cultured in a medium composed of Neurobasal A, Glutamax, pen/strep and B27. After 2 DIV the cells appears like neurospheres ans they started to show processes and get connected to each other. why neurosphere like structures appear in primary cortical neuronal culture?

just line, company etc

Hello,

I'm having purity issues with my DNA that I extract from mice tails. Here's the protocol I use . I run a PCR after that to identify knock out mice, but my gel had a lot of smearing. so i decided to do ethanol precipitation and it still isn't great, as you can see in the file below.

I really am not sure how to approach this, the PCR has to be definitive because subsequent experiments depend on the KO genotype.

I would appreciate any suggestion, because i can't keep asking for tail cuts :(

We are doing primary osteoblast culture from mouse calvaria, but we always get those very big cells as shown in the red cycle of the picture, which takes a lot of areas and blocks the real osteoblasts to grow. Does anyone know what is those cells and how to get rid of them from the beginning? Thanks a lot!

I injected AAV through mouse tail vein, but after one week when I sacrifice the mouse and extract the protein from heart, liver, spleen, lung, kidney and small intestine, I didn't detect the protein i injected. Is there something wrong? How to make sure my injection is successful?

We need to use Doxycicline I.P injections in an immunosuppresed mouse model, but when I prepare the solution the pH drops (both in water and PBS), being unsuitable to use in mice.

Do someone can tell me which buffer use?

Thank you

I am seeking guidance on dissolving hydrochlorothiazide (HCTZ) for intraperitoneal administration to mice. HCTZ readily dissolves in dimethyl sulfoxide (DMSO), but I have encountered precipitation issues when attempting to dilute it with saline or Hank's Balanced Salt Solution (HBSS). Are there alternative methods or solvents that can facilitate the dissolution of HCTZ while maintaining its suitability for intraperitoneal administration in mice? Any insights or recommendations would be greatly appreciated. Thank

Usually I culture mouse T cell with RPMI media and the mouse EC cell lines(C166---adherent cells) always culture with DMEM. I want to establish a in-vitro transendothelial migration model that mouse T cells migrate through C166 monolayer. So I want to know if DMEM medium affect mouse T cells activity and function. Could I use DMEM medium to culture mouse T cells in order to adapt the DMEM culture environment .

Hi, im looking for mice osteoblast cell line ( not MC3T3) for in vitro work. any suggestions?

I am wondering if I can make a genetically modified mouse to have breast cancer. I saw that mouse model named MMTV-ErbB2 can spontaneously develop breast cancer, but is breeding with these mice the only way to obtain breast cancer mice model with the gene modification of my interest?

Are there any easier ways like just administering some chemicals?

Hello,

i have to transport some primary mouse hepatocytes in some 6 wells plates at 37 degrees celsius ideally. It's going to be a 45 min drive approximately.

Does anyone have any ideas or recommendations about how to transport the plates without inflicting lot of damages to the cells ?

My study focuses on developing a mouse model for the type II diabetes, narrowing in on a condition resembling chronic, late-stage diabetes. As such, blood glucose levels would need to be measured consistently over the course of the 16-week experimental period. I will be using C57BL/6 mice. With the welfare of the animals at the fore, I would like to know if it is safe to perform fasting blood glucose tests on a weekly basis. Will it overstress the animals, and perhaps serve as a confound the overall development of the condition?

I need to get cerebrospinal fluid from freely moving/behaving mice without stressing them. How would you do that?

I'm experimenting on Swiss Albino mice and my goal is to make them obese. I don't know if there are any criteria to evaluate whether I have successfully grown fat or not? I read a few articles, they said that the weight of the fat was 40% higher than the control as success. Pls help me and thanks a lot.

I am interested in a mouse model of a controlled cortical impact that would mimic sports concussions by creating a mild TBI without any incisions, but I'd like to try and create specific coordinates and target specific brain areas. I figured I could use the eye as a landmark to find Bregma, but I can't seem to find an atlas for the head like you'd find for the brain or skull. Is there a reference for the average mouse skull size with measurements like the mouse brain atlas, or another effective landmark for locating Bregma without creating an incision?

How to prevent the death of mice after streptozotocin injection in the cerebral ventricles of mice for Alzheimer's disease induce?

I need to isolate live bacteria from mouse tissues. I have the "FastPrep-24™ 5G bead beating grinder and lysis system" and I will be using sterile tubes pre-filled with 3.0mm Zirconium beads. Tissue homogenates will be plated and assayed for bacteria growth. Has anyone done this and perhaps have a protocol or suggestions? I am concerned about bead beater settings, since I would not want to damage the bacteria with settings that might be too harsh.

Thank you!

Hello, I am now doing the HE staining of mouse skin paraffin section, but my slices always looks like not being well stained by eosin. I tried different staining time and use the dyes from different company, but the result always the same.

The skin was cut at 6µm,and before deparaffinization I put the slide in the oven at 56°C for 1 hour. This is my staining protocol: 2x7min xylene, 2x10min 100% ethanol, 5min 90% ethanol, 5min 70% ethanol, 5min ddwater. 10min hematoxylin, 2sec 0.1% HCl, 5min tap water, 3min eosin, 10sec tap water, 10sec ddwater, 10sec 70% ethanol.

The dermis seems different form others staining result, and I don't know why.

Please help me! Thanks!

The first one is my staining picture and the second is the brain HE staining using the same protocol. The last one is HE staining from the aticle "Protective Effect of Chitin Urocanate Nanofibers against Ultraviolet Radiation" .

We are trying to perform mouse liver biliary tree（bile duct) staining, is there a detailed protocol to follow? Any help is highly appreciated.

Warm Regards,

Liang

Hi, I am currently trying to isolate immune cells from mouse colon tissue for my project. For this purpose, I am using a protocol which includes ;

- Surgical removal of the colon tissue from mouse - Cleaning the tissue from surrounding fat and feces - Cutting the colon longitudinally and into small pieces (~4, 5 mm) - Incubating with 2 mM EDTA at 37C with mild shaking for 15 min x2 (removal of EDTA in between) - Washing the pieces in a strainer with 1X PBS x4 - Cutting the colon tissue into smaller pieces with a scissor - Digestion for 1 hour at 37C with digestion mix (1mg/mL Collegenase II, 2U/mL Dispase II, 80ug/mL DNAse I in DMEM with Glutamax) - 20 sec vortex - Quenching the rxn with 2 mM EDTA and washing with PBS/2%FCS - 10 sec vortex - Filtration (40 um) into new falcon to remove tissue - Centrifugation for 7 min, 1500 rpm.

At the end of this protocol, I always encounter formation of a slimy, white, powder like precipitate in some of my samples. These samples take quite some time to filter through 40 um filters and at the end, usually no cell pellet was observed in these tubes. I have previously tried to optimize the protocol by changing the concentration of digestion mix, digestion time, EDTA incubation time and washing the tissues after EDTA incubation with several ways, however none of my attempts stopped the formation of this precipitate. It appears to be happening randomly between my samples in every experiment irrespective of the genotype, treatment etc.

I couldn´t be able to pinpoint the source of error in my procedure, so my intention is to get the opinion of you, fellow scientists. Is there any other people present in this platform that also works with a similar protocol and maybe encounter similar problems? I am open to further discussion and suggestions.

It just came cross my mind - if matrigel is derived from mice, would there be an effect on the human organoids that we are culturing/generating? If so, how can we measure the changes? is there a human version of matrigel?

hello everyone，

I want to know the gene function in human cells. For example, I input some cell, and the datanase can tell me the function of genes in the cell, especially the role of positive regulation or negative regulation, like https://www.informatics.jax.org/ (mouse). I'm not sure if I made it clear, thank you so much for helping me.

We have injected GFP and td-tomato viral tracing injection in the mice brains to see axonal projections, and we are using tertiary butyl alcohol instead of ethanol for the dehydration process as well as xylene. But we are losing our fluorescence.

What we can do please suggest any other solution or any procedure.

So please, Thank you in advance.

I hope this will help us.

Note: We don't have cryostat with us.

Hi!

I have IP ed HA tag and Mouse IgG in two conditions of HUH7 cells. How come I see a band in the Mouse IgG lane around 78KD exactly the molecular weight of my desired protein that is HA tagged (even though I have tried an antibody for my western blot that is Rabbit so that I won't get cross reaction from same species that I did the IP with(which was mouse)). I don't see heavy and light chains.

Thanks everyone!

I'm gonna do IF in old mouse frozen sections to detect the pP53 and P21 level. The tissues were fixed by 4%PFA at RT for 30 min, 20um thick. The 2 antibodies are made from mouse, whcih caused the background signal is strong and there seemed no positive cell detected. I tested the antibodies in cell line by adding etoposide( to induce senescence).Both of them worked well. So any suggestions to help stain in the frozen sections? Many thanks!

I got this plasmid from Addgene (https://www.addgene.org/83481/) that I want to put into some mouse immortalized myeloid cells.

The basticidin resistance gene is driven by human phosphoglycerate kinase 1 promoter (hPGK). Will this be a problem?

I have nhe6 KO mice, confirmed via genotyping from their tail clips. I isolated BAT, iWAT, and gWAT from three controls and three KO mice. RNA isolation followed by cDNA preparation was performed from these isolated tissues. On running qPCR, I expected low ct values (high nhe6 expression) in controls and high ct values or undetermined ct values (may be?) for KO samples. However, shockingly! I got similar ct values (30-32) for both- controls and KO.

The questions here is-

Is it correct to perform qPCR on genomic knockouts? AND WHY?

My study aims at developing an animal model for type II diabetes. I don’t quite understand the power calculation, particularly in regards to the values I would substitute into the equation, as it relates to the development of the animal model, and not the treatment of an already developed model. How can I go about calculating the number of animals I require for my study to develop this model?

Keep these things in mind:

- the proposed model is for type II diabetes in combination with its microvascular complications

- I currently have two STZ dosage mechanisms: multiple low dose (MLD X 5 days) and single moderate dose (SMD)

- for the SMD and MLD groups, there are 3 doses per group, as well as 2 controls, for a total of 10 overall groups (6 subgroups will be treated with STZ and 4 control groups)

- in order to validate the disorder and the associated complications, I will be euthanising 3 animals from each experimental group, fortnightly for analysis.

I'm trying to measure OVA-specific IgE with a DIY ELISA, with an OVA coating. The issue I am facing is that since in the model I am using OVA-specific IgGs are produced in much higher concentrations than IgE, low dilutions of the serum get lower signal than higher dilutions. This reduces my signal significantly compared to my standard, which works great.

Would pre-incubating the sera on anti-IgG-coated plates help reduce that signal? And if so, has anybody done that before?

Thanks!

I am looking for VEGF A expression in mice placental lysates. However, according to the company (I use primary antibody from Abcam), I was supposed to see band 27kDa, however, I see 37kDa. They have shown 3 bands in mouse cell lines (27, 37 and 50 kDa). However, they say 27 kDa is a VEGF A protein. Could anybody please explain?

we have done a PCR for adhesion molecules comparing lung tissue from healthy mice to inflamed mice. surprisingly, the RNA expression in the inflamed mice is much lower than the healthy ones. I don't know how to interpret this, we are ruling out technical problems. Has anyone seen this before?

I am trying to find out parasite burden of spleen in Leishmania donovani infected mice using limiting dilution assay. I have plated in serial dilution and in many replicates but now I am confused as to how I can analyze the data.

Can someone please help?

Hi,

While we are doing genotyping of mice, we observed non-specific band which has main band size.

So we had 3 negative controls : (1) Taq only (2) Taq + primer (3) Taq +gDNA

Surprisingly, we got a 300bp~ band in (2) Taq + primer sample.

Is this a primer contamination?

Is it possible to have a 300bp~ band because of primer contamination?

Thanks in advance.

Sohee

Mice behavior after Tamoxifen injection: what does it look like?

Dear community, I will be very grateful for any clues about the issue. I was stacked with problems regarding mice's behavior immediately after an intraperitoenal injection of Tamoxifen.

Mice's behavior changes drastically after injection. They become much less active, sometimes with no movement at all; their eyes are squinted; some of them move only a couple of steps even after being touched by the experimenter. One interesting fact is that they sit separately, while normal mice are trying to get together. I observe such conditions even 24 hours after injection. In my previous experiment, I observed the same behavior after injection, and 13 of the 18 mice died. That was so frustrating. In my recent experiment, I used a completely new reagent, and I was extremely careful with the preparation of tamoxifen, including dosage and injection. I have even slightly reduced the injected amount, just by 0.005-0.01 ml. But still, the behavioral changes looked exactly the same. I am really perplexed because MANY articles use this concentration and dosage, and they refer to it as safe! One¹ of the articles even declares that "Over several years, we observed a mortality rate of 3.7% after three injections of TAM (n = 4.080, adult"mice)" — they injected 100 mg/ 1 kg for three consecutive days! I saw the articles about the influence of tamoxifen on some behaviors (anxiety-like, depression-like, locomotion, etc.), but I cannot find a detailed description of the behavioral phenotype immediately (during 48 hrs) after injection.

If anyone can share their experience, I will be grateful!

Short information:

1)

I need to stain mouse brain sections with c-fos and phospo s6 (which is rabbit). I'm looking for a c-fos ab (mouse) because I don't want to perform a multiple immunolabeling with primary ab originating from the same host species.

Hi, I am isolating primary mouse hepatocyte. The problem I am facing is sometimes I am getting very low viable cells (<50%). Sometimes I am getting really good number of viable cells (>80%). The problem is particularly occuring with one specific strain so this could be strain specific. But few other things that I would like to include. Whenever I am getting low cell viability although the liver gets pale in washing step and looks like digested in successive digestion step, but the liver does not inflate during the start of perfusion. Is it happening due to this? When I am getting good viable cells the liver generally inflate within seconds of perfusion. And also the liver looks clumpy even after it looks digested. But the clumpiness is not seen when I am getting good viable cells. Although I observed this thing in a particular strain I may not be right as well. If anything I am doing wrong. Please suggest.

I'm not sure why I'm getting a lot of non-specific staining from a rabbit secondary only control DAB stain. It looks like the pattern of staining is picking up blood vessels from my guess.

I quench using 1.5% H2O2 diluted in MeOH for 30 mins followed by permeabilization and blocking with 10% horse serum and 3% triton. Overnight incubation of antibody in 2% horse serum and 3% triton followed by incubation of secondary antibody for an hour and then ABC solution for 30min. All of these steps include 3x 5min washes in PBS in-between.

I perfuse the mice with PBS and then 4% PFA followed by incubation of PFA overnight. 30% sucrose is added to cryoprotect the brains.

I'm not sure if this is in artifact from the perfusions or if something is going wrong during the staining itself. Any help would be appreciative!!

I had to perform an LC-MS to check the post translaltional modifications of my histone isolated samples from mice brain. Can anyone let me know that how to perform desalting of my sample for LC-MS...????

Hi , I am looking for a full protocol for immunostaining of mouse pancreatic islets after isolation. I put them in RMPI after isolation. How can I prepare them for staining? and can I do costatining with two antibodies?

In order to study melanoma lung metastasis in aged hosts, we did RO injection of B16F10Luc2 melanoma cells (expressing firefly luciferase) into both young (16 weeks) and old (>2 years) mice. However, we noticed a fair amount of signals were coming from the eye area when doing IVIS on these mice meaning many of the cells injected were trapped in the eye and forming localized tumors instead of metastasizing to the lung. I wonder if this is a normal thing and if there are any ways to prevent this from happening.

What is the overall protein content in one mouse cardiomyocyte?

I use STZ 50mg/kg/day(ip) for 5 consecutive days to induce diabetes in mice. Solve STZ in 0.05M Citrat buffer pH 4.5. However, some mice died within 5 minutes after STZ injection. Autopsy results in mice revealed ascites. I have no idea why they die and produce lots of ascites. Does STZ cause ascites in mice?

I want to isolate mouse bone marrow neutrophils to study neutrophil extracelluar trap, but the vechicle group always activation.

We are currently working with MN9D cell line. MN9D cells are mouse midbrain that are Dopaminergic. We do several HPLC analysis on the MN9D cells for over 20 years and have always seen dopa and dopamine peaks. For the past month we have not seen any dopamine peaks in HPLC, but only dopa peaks, even in our control samples. We know it's not a HPLC problem because injected a dopamine standard and it shows nice sharp peak. We have kept all our conditions constant in terms of our cell culture method. Our Passages number are pretty low as well. I am not sure what is going on and why we don't see dopamine. Does anything have suggestions?

Could bis (cyclohexanone) oxaldihydrazone soluted with water to feed mice induce demyelination model?

Has anyone seen anything like this? The female Ornithodoros turicata engorged on a mouse and this is how I found it after taking off the animal. Normally this doesn't happen and nothing protrudes from the genital opening after a female tick is fed. My guess is this is a pathology of some kind. Perhaps because of the internal pressure generated by the sucking action the vestibular vagina prolapsed/everted somehow... Any ideas?

DuoSet ELISA

I have tried lowering the Tm, worked on human DNA but not on mice DNA. Tried optimizing denaturation temperatures too. Any suggestions?

Hello everyone,

I have the following question regarding the number of organoids in culture.

I performed a seeding assay with mouse tumor colorectal organoids. I seeded 2000 cells (dissociated from the organoids) and take pictures after 3 and 5 days. And then count the number of the experimental and control.

I observed a difference but the question is, Why is this possible? If I seed the same amount of cells and from this individual cells the organoids will grow, why the number of formed organoids can be different?

Some cells from a forming organoid scape and grow independently to form another organoid? Or some cells from the 2000 seeded cells do not grow and die?

All your comments are welcome

Thank you in advance.

Hi, currently in our lab theres a lot of transgenic mice model generation, wich means a lot of breeding and lot of genotyping. We are using tails, we lyse them ON and run a PCR and a gel. The problem is since we got up to anywhere from 20 to 100 tails a day and they have to be genotyped for up to 6 genes this method is proving to be very time consuming.

Im trying to find a streamlined method that dosent involve so much man hours to complete.

P.S : we just have una qPCR machine but 3 normal thermocyclers dedicated to genotyping.

Looking for a good STING/TMEM173 unconjugated, monoclonal antibody, reactive in mice for western blots, immunoprecipates, and potentially immunofluorescences. Ideally, I'm looking for antibodies for unphosphorylated and phosphorylated forms of STING/TMEM173.

Any recommendation for TXNIP western blot? Adult mouse cortex samples

I am planning to induce status epilepticus with a subconvulsive dose of pilocarpine. I have experience with the pilocarpine model but try to find out a subconvulsive dose.

I am wondering to block the synthesis or at least suppress cholesterol in a mice model and I was wondering how to do it and which are the best options to avoid using the knock out mice.

What would be the impact if I removed the teratoma and kept the mice alive?

I am running mouse amyloid beta 42 ELISA using the kit from Invitrogen. According the manufacturers guideline, the samples should be diluted so that the concentration of guanidine in final solution is <0.1M. Of note, the homogenization buffer is 5M Guanidine/50mM Tris. Even after diluting the sample to final guanidine concentration of 0.08M, I am seeing suppression of the standard curve. Has any one experienced this before? Thanks!

My research aims to produce a novel mouse model for T2DM and I have two dosage techniques to administer STZ in C57BL/6 mice: single high dose (SHD) and multiple low dose (MLD). Within these groups, in my study design I have divided my animals into three subgroups:

- SHD: 75 mg/kg, 100 mg/kg, and 150 mg/kg

- MLD: 40 mg/kg, 50 mg/kg, and 60 mg/kg, each for 5 days.

When it came to the MLD groups, I basically sandwiched the standardised protocol provided by DiaComp of 50 mg/kg dose of STZ with a dose 10 mg/kg higher and lower. However, the ethics committee is requesting a substantiation for this and thus I would like to know if what I did is correct, and if there's literature to back this up, or if I should change my MLD dosage groups altogether. Please assist. Bear in mind, the model aims to assess the major microvascular complications, and thus the diabetic condition should mimic the later stages of the disease.

Thank you.

I am working on simple Y maze these days. I observed that most of my disease mice didn't show any spontaneous alteration in the experiment? However, some of the mice show some alteration. Could anyone suggest how to represent these data. because when I combined them together data has high SD.

I am trying to mesuare intracellular ROS with DCF dye in isolated lung cells from mice that have been treated with LPS for three days. The problem is that I have lower ROS production in LPS-treated mice than in naive mice, when it should be higher. We incubate the cells with 10 uM DHCF for 30 min at 37 degrees, then we wash the cells and mesuare the fluorescence by flow cytometry. Maybe there is another way to do it or there is something happening with the mitochondria? Maybe LPS induce mitochondria extrusion from the cell and we can not detect fluorescence?

I have an idea on how to dissect motor cortex from mouse and I'd like to find an article or a book, does anyone have some piece of information?

Hi everyone,

I would like your opinion on using the complete() function of the mice package on R.

Theoretically, after multiple imputations, analyses should be performed on each imputed dataset, and then the results of the analyses should be pooled (see attached diagram).

However, I consider the complete() function. In summary, it permits generating a unique final dataset using the results of multiple imputations previously performed with the mice() function. This strategy is easy, "inexpensive," and allows us to manipulate only one dataset.

This is a concrete example of the usefulness of this strategy. I am conducting mixed-methods research in which I want to interview some participants after analyzing their responses to my survey. If my respondent John Doe did not answer to an item of a scale, I would risk having 5 plausible answers from John Doe after multiple imputations (if m=5, or 20 plausible responses if m=20, etc.). However, the complete() function will summarize the different estimates into one dataset (instead of 5, or 20, etc.). Basically, during an interview, I will be able to question John Doe based on his scale score computed with NA replacement. So I lose precision, but gain in ability to exploit the answers.

However, this approach seems problematic, as the literature does not support it well. In fact, except for this paper by van Buuren et al. (2011, cf. section 5.2), I cannot find any source that supports this approach:

van Buuren, S., & Groothuis-Oudshoorn, K. (2011). mice: Multivariate Imputation by Chained Equations in R. Journal of Statistical Software, 45(3), 1-67. https://doi.org/10.18637/jss.v045.i03

Well, I'm stuck between a rock (a more rigorous approach, i.e. the pooling) and a hard place (a more practical approach, i.e. the complete() function). What do you think?

Hope to read you (and my apologies for my broken English)

FM

I performed a western blot using different tissues from the mouse(brain,lung,liver,spleen,small intestine, colon, kidney, testis). However, I observed a significant amount of background on the membrane. Why is there more background in the tissues during the western blot, but none in the cell line? What are the differences between them, and how can I reduce the background in tissue samples in the western blot?

For protein isolation, I used RIPA buffer containing a 1X protease inhibitor. I homogenized the sample using a rotor-stator homogenizer and then centrifuged it at maximum speed at 4°C for 30 minutes.

For western blot sample preperation, I diluted samples 50 ug in 16 ul RIPA buffer containing 1X protease inhibitor. I added loading dye with 0.2M DTT then I boiled it at 85 °C for 5 min.

I performed 2 hours blocking step and then incubated overnight with the primary antibody ( I prepared with blocking buffer)

I used primary ab 1:1000 for anti-A protein(from rabbit) and 1:6000 for anti-actin(from mouse) For secondary ab, I used 1:15000 anti-rabbit and 1:15000 anti mouse.

To reduce the background, I checked the antibodies by performing three separate western blots. In the first western blot, I omitted the primary antibody to test the specificity of the secondary antibody. In the second western blot, I used a lower concentration of the primary antibody: 1:2000 for anti-A protein (from rabbit) and 1:6000 for anti-actin (from mouse).In the third experiment, I used tubulin, but I didn't observe any difference.

Can anyone share tissue processing protocol for mice brain ?

I am working to develop a Mac Elisa protocol that is able to capture the level of Immunoglobulin M (IgM) in mice infected with fungus T. marneffei. I've been mainly running into trouble with false positives from unspecific binding, but I've also had the opposite problem where I got a negative signal from a known positive control. As such, I've been tinkering with the concentration and incubation time of the reagents, including running a couple of concentration grids, but haven't found much success. I am wondering if anyone on here has much experience running or troubleshooting Mac Elisas, and has anything to suggest trying out! Thanks!

Current protocol:

Step 1 (Coating): Anti-mouse IgM polyclonal antibody (5ug/ml), 100ul overnight @ 4 C

Step 2 (Blocking): Blocking buffer, 200ul for 2 hours at room temperature

Step 3 (Sample incubation): Mouse plasma (diluted 1:20), 100ul for 1 hour at 37 C

Step 4 (Antigen): Detect anti-MP1P mouse-IgM with Recombinant MP1P (0.1 ug/ml), 100ul for 1 hour at 37 C

Step 5: Detect immune-complex with Biotinylated anti-MP1P mouse monoclonal antibody (MAb) (1:1000), 100 ul for 1 hour at 37 C

Step 6: Detect biotinylated anti-Mp1p mouse MAb with Stretavidin-HRP (1:2000), 100ul for 30 min at room temperature in the dark

Step 7: Detect HRP with TMB, 10 min at room temperature in the dark

Step 8: Stop activity of TMB with HCL 0.3M

Hello! I am planning to perform the Object Location Memory Test for mice. I have done the Novel Object Recognition Test before with a protocol my former lab used (four days in total: 1 day of habituation, 2 days of training (same objects) and last day of test (switching one object for a new one).

Which protocol would you recommend for the OLMT? I have seen different ones but I wonder if there is a main one people usually follow.

THank you!

Can anyone share the tissue processing protocol for SEM/TEM of tissue slides of mice?

Hello -- does any one have any recommendations for medication delivery vehicles (food items) that have worked well for C57BL6/J mice. The drugs we plan to use are somewhat bitter. Our study also needs to avoid foods with fat so we cannot use nut butters, chocolate, or dairy products.

Thanks in advance.

Do I understand correctly that heterozygous Pmel mice have twice as less cells that carry anti-gp100 TCR? This should be right because of the allelic exclusion. If so, can one combine both homo and hetero mice in one experiment (if doesn't have enough homo) just by doubling the number of transferred hetero cells?

AS the cd4+ T cells of OT-II mice are specific for chicken ovalbumin 323-339 peptide,OVA protein plus Aluminum hydroxide adjuvant can lead to the specific proliferation of Th cells，why some articles also plus LPS to induce immune? Does LPS induce the non-specific proliferation of Th cells?

RNA from the mouse skin tissue.