Adipose Tissue - Science topic

Sucralose is a synthetic sweetener that is approximately 600 times sweeter than sucrose. It is made by partial chlorination (replacing OH groups with Cl) of sucrose. Sucralose is very heat stable and can be obtained in powdered form for use in baking applications. It has numerous food applications, including candy, soft drinks, and sweet syrups. Sucralose is available in granulated and powdered form. Granulated sucralose is mixed with fillers to provide a measure for measure substitution with table sugar (sucrose). The powdered form of sucralose contains 90% bulking agents such as maltodextrin, that are a metabolizable form of carbohydrate. A 50/50 mixture of sucrose and sucralose, plus a bulking agent, is available for baking applications. This mixture reduces caloric content and enables browning reactions in baking applications. The safety of sucralose has been extensively investigated and is considered safe by FDA, the Joint FAO/WHO Expert Committee Report on Food Additives, and the European Union's Scientific Committee on Food. The ADI for sucralose is 5 mg per kg of body weight which equates to approximately 390 mg for a 175 lb person.

Hi Amanda Madureira,

Check this article: They worked with PAS on embryonic adipose tissue cryosections.

However, PAS is not specific for glycogen, since it stain other molucules too, as stated here

Good luck!

Greetings!

You can find the required formula for fat mass percentage and fat mass index based on BMI, age and sex on the following link:

Also, in supplementary materials, you can find Excel table with calculations, you only need to enter your numbers.

Hope it is helpful.

There are several methods that can be used to measure body fat percentage and determine whether a person is within a healthy range. Some of the most common methods include:

It is important to note that no single method is considered to be the "gold standard" for measuring body fat percentage, and different methods may produce slightly different results. Therefore, it is generally recommended to use multiple methods in combination to get a more accurate assessment of body fat percentage.

To determine whether a person is within a healthy range of body fat percentage, it is generally recommended to use guidelines from organizations such as the World Health Organization (WHO) or the American Council on Exercise (ACE). These guidelines provide ranges for different age and gender groups, and can help to identify whether a person is at an increased risk for health problems due to excess body fat.

Hello Syeda Ali

Choosing an appropriate technique depends largely on the required purity, yield, and speed, as well as on cell viability and functionality after extraction.

Density gradient centrifugation has limited throughput and specificity. The two most common methods used are MACS and FACS which improve on these parameters in different ways.

The main difference between MACS and FACS is that MACS provides bulk separation of cells, whereas FACS is based on single-cell sorting. Also, MACS has a higher throughput of the two techniques, while FACS utilizes more parameters and, thus, is more specific.

So, from the techniques that you have mentioned, I would suggest you choose either magnetic bead–conjugated antibody cell isolation or FACS.

Best.

Indeed, that is what I'm saying Sebastian, the color intensity changes at high concentrations are no longer proportional to changes in protein. This does represent a loss of sensitivity by definition at concentrations >2 mg/mL

Hello Syeda Ali

Macrophages can be categorized into two main types: M1 and M2 macrophages. The M1 type, referred to as classically-activated macrophages, are activated by pathogen invasion and play a large role in the immune response to foreign pathogens such as bacteria.

The M2 type, referred to as alternatively-activated macrophages, play a role in wound healing and tissue repair, and have an anti-inflammatory role.

M2 macrophages can be further divided into M2a, M2b, M2c, and M2d subcategories. These macrophages differ in their cell surface markers, secreted cytokines, and biological functions.

So, depending upon the type of macrophage you can select the markers.

For instance,

CD68 and CD11b are total markers of macrophages.

M1 and M2 macrophages have specific markers.

M1 Macrophage Marker

For M1 you may choose CD80, CD86, CD64, CD16 and CD32 as markers. In addition, the expression of nitric oxide synthase (iNOS) in M1 can also serve as phenotypic marker.

M2 Macrophage Marker

CD163 and CD206 are major markers for the identification of M2 macrophages. Related surface markers for M2-type cells also contain CD68. Compared with marker CD68, CD163 is more selective to macrophages, so CD163 can be used as a highly specific marker for M2-type macrophages.

M2 macrophages are sub grouped into M2a, M2b, M2c, and M2d.

M2a macrophages

M2a are activated by IL-4 or IL-13. M2a macrophages lead to the increased expression of IL-10, TGF-β, CCL17, CCL18, and CCL22. These macrophages enhance the endocytic activity, promote cell growth and tissue repairing.

M2b macrophages

M2b are activated by immune complex, Toll-like receptor (TLR) ligands and IL-1 receptor ligands, and they can express IL-6, IL-10, TNF-α, and IL-1β. Based on the expression profiles of cytokines and chemokines, M2b macrophages regulate the breadth and depth of immune responses and inflammatory reactions.

M2c macrophages

M2c also known as inactivated macrophages, are induced by glucocorticoids, IL-10 and TGF-β. These cells secrete IL-10, TGF-β, CCL16, and CCL18 and play crucial roles in the phagocytosis of apoptotic cells process.

M2d macrophages

M2d can be induced by the TLR antagonists, and M2d macrophages express IL-10, IL-12, TGF-beta and TNF-α.

Best.

This looks ECM (top picture). Since you 've ruled out potential contamination, the other structures are likely medium components that precipitate and aggregate for some reason..

You should make the Frozen room colder to -30 because of high concentration of Lipid and also you can cut your tissues with thicker trims like 8 mm, however, some other ways like embedding your tissues with Cold Nitrogen instead of Frozen glue would be more suitable.

I find a lot of people don't realise quite how dilute cDNA can afford to be.

cDNA synthesis buffer itself is inhibitory to PCR (it contains DTT, for example), and you can also inhibit your reaction by saturating the system with too much target (or too much non-specific target).

In almost all cases, cDNA should be diluted prior to qPCR.

I typically dilute all my cDNA 1/20 (so for a 20ul prep I add 380ul water, giving me 400ul of final cDNA), and using 2ul of this per reaction is _plenty_: it allows me to reliably detect even low abundance genes.

For reference I use ~1600ng of RNA in a 20ul cDNA synthesis reaction, so 2ul of diluted stock is ~8ng of cDNA, assuming 1:1 conversion.

In your case it may be nothing to do with the lipids and everything to do with just...generally using too much cDNA.

When running dilution curves of cDNA historically, I expect the extremes at either end to be unreliable: with too much cDNA you have all the issues noted above, and with too little cDNA you have stochastic effects and thus highly variable data. This is FINE. The whole point of a dilution series of cDNA is to establish the range over which your reaction is linear and trustworthy.

Find that range, use it, don't worry about the extremes.

How about your positive and negative control? Is that valid?

If Beta-actin also didn't amplified, it is either there is inhibitor in the master mix or samples or there is no cDNA inside the sample.

Beware of ethanol or alcohol, which can serve as the source of inhibition. If your positive also without amplification, it might be the problem of master mix or qPCR protocol.

Thanks for the info. Glucose is metabolized immediately, so you should look at the 2-DG uptake.

This method is easiest and most reliable using RI labeled 2-DG, but colorimetric and chemiluminescence detection is also available.

(However, I only have experience with RI-based method.)

*Colorimetric method

https://www.cosmobiousa.com/products/2-deoxyglucose-2dg-uptake-measurement-kit

*Chemiluminescence

Glucose Uptake-Glo™ Assay

Please carefully examine the methods.

Good luck!

perturbations and that body fat distribution is an important determinant of obesity-related complications. Individuals with increased upper-body adiposity are disproportionately burdened by obesity-related diseases, compared to lower-body obese individuals. Thus, it is paramount that studies continue to elucidate the pathways linking various adipose pads and depots in relation to health and disease, as well as the mechanistic underpinnings dictating how body fat is distributed in order to answer fundamental questions. Rodents are commonly used to model features of human metabolism and obesity, yet it is unclear to what extent rodent fat pads are a suitable model of human fat depots. Here, we have highlighted examples of both shared and divergent traits among rodent fat pads and human fat depots. Given some of the stark differences in adipose tissue location and function among species, we urge careful consideration in experimental design and interpretation when attempting to draw definitive parallels between rodent fat pads and human fat depots.

Thanks for the tip. Until now, I have not fixed the tissue in succrose before embedding.

I will try that and also cut in colder temperatures:)

Kind regards

Jasper

Dear Parhesh Kumar , I haven't personally tried it, but my guess is that is should be possible, since cryofixation methods are applicable to adipose tissue. You can maybe if someone has conducted it even if it was with a different tissue to check whether they performed the following steps with different staining conditions. Good luck!

Hi, arigo has an NLRP3 Inflammasome Antibody Panel which includs antibodies to NALP3, ASC, and caspase1 with small package. Please refer to the detail at https://www.arigobio.com/NLRP3-Inflammasome-Antibody-Panel-ARG30331.html.

Also check https://www.frontiersin.org/articles/599277

Hi Archana, yes, our medium was already supplemented with L-glutamine sodium pyruvate, but still, the cells remained stressed and fragile. In fact, there are very few in the culture bottle now and we don't know if we will be able to save it.

Years ago, before MSC were identified, I used to culture murine bone marrow stromal cells. In that culture system, the medium was RPMI 1640 + 5% FBS - NOT heat inactivated. Since then, I have worked with human MSC from bone marrow using the spicule fraction, and for that work, I used DMEM - low glucose/ 1 gm per liter + 10% FBS (also not heat inactivated).

Some key points I have found are 1) seeding density is important - at least for bone marrow stromal cells. They produce an autocrine factor [which turns out to be M-CSF] and at too low a cell density, they do not proliferate.

2) Passage before the culture reaches confluence is crucial - particularly for MSC. MSC undergo contact inhibition - a major decrease in gene expression - and once they are in that state, they grow painfully slowly. This is likely where bFGF comes in - as noted by Wondong Yu.

3) Use FBS that has not been heat-inactivated. The grade of the FBS seems to be important - not all products work for MSC cultures. You need to test suppliers. We have had good results with FBS from Sigma - catalog # F2442.

4) Using low glucose medium is important - at high glucose, you get an outgrowth of adipocytes. DMEM comes at either 1 gm per liter or 4.5 gm per liter. Use the 1 gm/liter DMEM.

One other point to note - DMEM/Ham's F12 at 1:1 is essentially the same as Iscove's Modified Dulbecco's Medium {IMDM].

Dear Ana Beatriz dos Anjos Souza ,

These fluorescent lipophilic dyes, which partition into the non-polar lipid droplet core, are useful lipid droplet markers. BODIPY™ 493/503 4,4-DIFLUORO-1,3,5,7,8-PE 10 MG is according to: https://www.thermofisher.com/order/catalog/product/D3922#/D3922A dye (so not an antibody) that is described as ”With its nonpolar structure and long-wavelength absorption and fluorescence, BODIPY® 493/503 can be used as a stain for neutral lipids and as a tracer for oil and other nonpolar lipids”.

4,4-DIFLUORO-1,3,5,7-TETR 5 MG (BODIPY™ 505/515 (4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene)) https://www.thermofisher.com/order/catalog/product/D3921#/D3921is described as ”With its nonpolar structure and long-wavelength absorption and fluorescence, BODIPY® 505/515 can be used as a stain for neutral lipids and as a tracer for oil and other nonpolar lipids.” Indeed, the same. Looking at the structures the difference is one methyl group.

Perhaps the following papers is of interest to you, it discusses the (possible) advantages in terms of spectral features between different dyes:

Best regards.

Hi Kim,

I am not working on SVF cells. But, reading your complaint about low cell yield, I guess the reason might be a simple issue. Here are some suggestions:

1. Make sure you grow enough number of cells. 6-well has a small surface area, and you may get quite low yield in the end. If cell size is relatively large, they may look confluent under microscope, but the number might be much smaller than you expect when you count them.

2. Make sure majority of cells detach from plate after digestion with trypsin. You can adjust time to ensure maximum cell detachment but no more than 5min.

3. Centrifuge cells at a slightly higher speed or longer at your original speed. This may increase your cell yield in case some cells didn’t precipitate well.

4. If you are using fixed-angled centrifuge rotor, your cells may stick to tube walls and you may loose them during suction. If that’s the case, you can switch to swinging rotor.

5. When you have low cell yield, you should be extra careful not come too close to pellet with suction tip. Just leave some media above the pellet.

These would possibly increase your cell yield. Good luck!

Hi Anna, are you trying to label the lipid, or the proteins sequestering the lipids? For labeling mature droplets, antibodies targetimg perilipin would work. For labeling (or imaging) the neutral lipid, bodipy can be used.

Also check https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214029

well these are adipocytes for sure !

Hi, you can check those answers as well.

Good luck

Also check https://rgd.mcw.edu/rgdweb/ontology/annot.html?acc_id=CMO:0000105&species=All

There is a number of issues that may occur with your measurements. Uneven loading due to incorrect measurements of total protein is a common problem for every researcher using WB. Quick solution for your experiment is to quantify the bands using image J, calculate the percentage, and then use it as the correcting factor for your initial loading values (i.e if sample 1 is 100%, but sample 2: 150% multiply sample 2 total loading to 0.5) and rerun the gel. In terms of fixing your quantification process, it is hard to say what exactly may be wrong as there are many factors, try to use update your standard curves.

See here: https://pubmed.ncbi.nlm.nih.gov/18281723/.

These drugs belong to the chemical class of retinoids, so I suggest you to consult scientific literature, you will find many protocols for extraction/purification of retinoids and vit A-related compounds from adipose tissues (e.g. https://doi.org/10.1002/neu.20243; https://doi.org/10.1016/S0003-2697(02)00662-0 ).

You can try a liquid-liquid extration. Make sure your aqueous phase is acidic (Perchloric acid pH 1.0) and I also like to deoxygenate before with nitrogen stream. After the extraction, you can perform either direct Electrochemical measurements or HPLC-EC or HPLC-UV.

Good luck!

Hi, maybe you have resolve your problem but I think you have to improve some gel condition, and liver samples preparation.

Did you make coomassie on gel, out of curiosity?

The best and only adipose tissue protein extraction kit is the Minute™ Total Protein Extraction Kit for Adipose Tissues/Cultured Adipocytes. It uses a strong cell lysis buffer and a patented spin column filter technology. Results give a complete protein profile and high yield unlike RIPA buffer. https://inventbiotech.com/products/minute-total-protein-extraction-kit-for-adipose-tissues-cultured-adipocytes?_pos=1&_sid=4dc367ec1&_ss=r

You should rather use 10% neutral buffered formalin as a fixative. For effective and early penetration in tissues fixative can be directly injected through vascular system so that it reaches tissue fast and fixation starts immediately. Even thereafter when you collect and keep tissue in fixative it needs minimum 24-48 hrs to get fixed properly (it depends on thickness of tissue collected)

Hi Monika,

Maybe it's too late you already have the answer but I experience it also and I was surprised! :) I believe it can also help other people in the future :) When you try to isolate total RNA from the adipose tissue I recommend you to remove fat layer after homogenization. Normally, if you experienced 4 phase after chloroform it means that you used quite big amount of tissue and there was a lot of fat layer. So, it is important to remove fat layer by pipet as much as you can before adding chloroform. To achieve this separation correctly, you should do a centrifugation right after homogenization process. 2-3 mins at max speed. I'm using 200ul tips to remove fat layer and it is working so well! Rest of the protocol same than regular RNA isolation procedure.

Good luck Monika Nanda and others.. :)

It depends on the collagenase activity reported by the manufacturer.

If for example the activity was ≥ 0.18 U/mg and you wanted a 0.3 U/mL stock the calculation would be:

(1000mg) X (0.18 U/mg) / (0.3 U/mL) = 600 mL

This stock can be made in in all buffers which are commonly used for cell isolation.

Thank you very much, i will try that!

Hello Luca,

The nerve impingement could be done -among other reasons- by the fatty tissue embedded within the lumbar fascia. Little is known about the structural changes that the deep fatty tissue can undergo. For exemple, local hyperplasia of the deep fatty tissue could cause cluneal neuropathy. Local hyperplasia palpable as nodules in the lumbar region have been called "back mice" and have been related to chronic low back pain.

Thanks,

Marta

Hi Jiulia, from your pics it looks like you have a heterogeneous population with senescent cells. But they do have fibroblast-like morphology! I would add 10-20ng/ml of FGF-2 to help... Anyway you should obtain a homogeneous population after 2-3 passages in culture. Which passage are you cells in the figures?

Best,

Yuri

Good day,

As other tissues. Fix for 24-48 h in 10% buffered solution of formaldehyde, then make parafin embibition by standart protocol with isopropanol or ethyl alcohol, and then get parafin blocks. It is in brief. Specific preparation of adipose tissue needed in cases of special stains like sudan III or kongo rot, when you need to visualise the fats.

Hi,

you can try gentleMACS™ from Myltenyi Biotec.

I like to add my view in respect of the answer given by Rasha Shakir Nima.

It is not true to suppose that any given pair of variables are linearly related so that any given set of data on a pair of variables can be represented by linear regression line.

Therefore , the degree of regression for the given set of data is to be determined first. This can be determined by testing the significance of regression coefficients (starting from linear regression coefficient to the regression coefficients of higher order).

After determining the degree of regression, the corresponding regression analysis is required to be performed.

If your data are distributed in a Gaussian way (normal distribution), then the cut off will be the mean+2SD. However, and to be more precise, you can create a 2*2 table, from which you can calculate the performance indicators of your test- mainly the sensitivity and specificity- from which you can construct a Receiver Operator Characteristic (ROC) curve where your tests' cut off shall be the point which possess, theoretically, the maximum sensitivity and specificity values.

Best wishes

hi paper,

you may coupling your macrophage staining with anti myosin heavy chain or perilipin immunostaining to mark fibers or adipocytes, respectively. Alternatively, you can perform a hematoxylin and eosin stain. In this case, adipocytes appear as white vacuolar unstained cells while fibers will be stained in red

Dear Alicia Bibiana,

There is a reference developed in 2006 by the Epinut research group from the Complutense University of Madrid but it is based on Spanish children. It provides percentiles by sex and age based on the 4 basic skinfolds and the equations from Durning & Womersley, Siri, and Slaughter. Here is the link to the article: https://www.analesdepediatria.org/es-pdf-13090892

Besides, there is a classical classification proposed by Westrate and Deuremberg in 1989 in where they stated that the presence of obesity in prepubescent children exists when the %body fat obtained by anthropometry is greater than 30% without distinction of age. This is the link to the article: https://academic.oup.com/ajcn/article-abstract/50/5/1104/4695440

I hope this could be useful for you. If anyone knows any other reference I'd also appreciate reading it.

Best wishes!

Jana Tumova Thanks Jana!

Hi Joao, The adipose tissue is very delicate and sections are difficult to stretch without damaging them. I would suggest that you prepare a 10% alcohol (ethanol or IMS) in distilled water and pipette a large droplet of this solution on a positively charged slide (if you wish to collect serial sections - prepare a row of such slides). Place the adipose tissue section on the surface of the droplet. The surface tension of the droplet unfurls creases of the section and that allows for the next step - careful lowering of the slide into a water-bath (keep the temperature above 45 deg C, but you need to experiment to find the best temperature which gives you enough time for collecting the section without rushing (depends on your thermostat and the type of wax you are using). Watch the wax becoming more translucent and immediately collect it on the slide.

Until you are used to this method - it would be better to collect one section at a time as it is easy to miss the best moment for collect a section from the water-bath.

Good luck with your project.

C. Juneja Chloe Henderson

Best variant is to fix tissue in big volume (over 20-25 volumes of tissue) of 4% phosfate buffer formalin (pH over 7.4) for 24-48 hours and then do histological embibition of tissues. Better leave parafin blocks before winter breaks.

Ethanol for fixation is not a good idea especially for a long period. Ethanol dries tissues and H/E stained tissues will be with artifacts.

Dear Dr/ Lee

I appreciate your reply. Would you please provide me a reference for 2^-dCT?

Thanks,

I did a similar thing in the past, we transplanted stem cells tagged with dapi (there is a version sterile for cells) and we stained the cells we put inside, the only draw back is that fades after a few days because the cells replicate and the dna (stained) becomes half at every reproduction cycle. If is for a short time you will be able to use this technique, again you can also use iodine red for the same purpose

TCIA Collections: TCIA is a service which de-identifies and hosts a large archive of medical images of cancer accessible for public download. The data are organized as “collections”; typically patients’ imaging related by a common disease (e.g. lung cancer), image modality or type (MRI, CT, digital histopathology, etc) or research focus. DICOM is the primary file format used by TCIA for radiology imaging. Supporting data related to the images such as patient outcomes, treatment details, genomics and expert analyses are also provided when available.

https://www.cancerimagingarchive.net/

Hi Adil

this SNP is far away the 5'UTR of the TMEM18 gene (around 19kb) and do not show any significant flanking sequence to explain its association with obesity (see attach). Thus this SNP seems to be more a marker for the disease than the the real causing variant. nothing for the moment, but still to be elucidated.

fred

Hello, Yongyao Wang

Morphology preservation depends on your freezing method.

If you use the cryogenic method of immobilization,

such as high pressure freezing,

sample should be preserved perfectly.

Otherwise,

liquid nitrogen plunging may cause severely ice crystal to damage your organelle in the ultrastructure of cells.

But you never know what will happen in EM world,

just do it, you’ll get more than you loss.

FYI

Wolf

Hello, here is a paper which my colleague found very useful and it has a nice protocol & shows the gating strategy

You should not need to use Matrigel when injecting into the mammary fat pad - as others have said, 4T1 will grow almost anywhere without the additional ECM support. Depending on your experiment, matrigel can potentially confound your analysis. It can be difficult to determine that you have implanted into the mammary fat pad rather than sub-cutaneously near the fat pad, since the cells grow equally well in either site.

I suggest that the hypolipidemic effect of gerciana extract be conducted using a model as well as LD50 before trial on human.

I had similar observations many years ago in C3H mice. There, our pathologists told me that the ascites was a consequence of injection-associated lipoma. We then moved to tumour induction subcutaneously in the legs of the mice.

W.

Thank you for your help Dongdong Xiao

How do you convert NOAEL in ppm to mg / kg / day?

Would anyone have any reference?

Are you evaluating how many viable cells are there before plating the cells? Also, you maybe should seed at a high concentrations, since many cells will die or are already dead, thus, it might be a good idea to concentrate their proportion.

Even so, If the number of cells after the extraction is too low, It might be a good idea to check that you are not digesting the tissue in collagenase for too long or that you are doing the protocol at a good pace.

Please check

1. Badaut J, Hirt L, Granziera C, Bogousslavsky J, Magistretti PJ, Regli L.Astrocyte-specific expression of aquaporin-9 in mouse brain is increased after transient focal cerebral ischemia. J Cereb Blood Flow Metab. 2001 May;21(5):477-82.

2. Hui Liu; Mei Yang; Guo-ping Qiu, Fei ZhuoI; Wei-hua Yu; Shan-quan Sun; Yun Xiu.Aquaporin 9 in rat brain after severe traumatic brain injury. Arq. Neuro-Psiquiatr. vol.70 no.3 São Paulo Mar. 2012

3. Jerome BadautLuca RegliLuca Regli. Badaut J, Regli LDistribution and possible roles of aquaporin 9 in the brain. Neuroscience.2004 129:971-981

4. Badaut J, Regli L. Distribution and possible roles of aquaporin 9 in the brain. Neuroscience. 2004;129(4):971-81.

Yeah! I agree with Ramos, 1: 10 is the standard ratio for tissue and Trizol in RNA extraction. With my experience, Fat or triglycerides will inhibit the quality of cDNA!

Why do you not want to use the radiological method? It is very sensitive and is well characterized. I think that if I were going to measure adenylate cyclase activity without P-32, I would try this method. It uses inhibition of luciferase as ATP is depleted.

Hi, what's the method you used in the last?

When the dimples of Venus are disappearing ( the Michaelis rhomb , used as marker by obstetrics ) where in modern times pretty womans are showing the sexy antlers tattoo, we have an indication for tissue swelling in this aea. This symptom often is connected with fibromyalgia in the lower quadrants, and the kidney meridians are irritated. This is caused by an entrapment of the nerves behind the inner ankles ( ! ). Then the diagnostics for Fibromyalgiasyndrom have to be started, as described in two papers from the Temple University, Philadelphia. After decompression and neurolysis of the entrapped little peripheral nerves the symptoms are disappearing and the fibro also. ( My surgical experience since 1998 - 3 patients the week ). Peers aren‘t allowing publications of method and results. Isn‘t „scientific“. Painfree patients are of no interest. It‘s strange, but the typical indicator for a very effective New Method......

following

Hi Jakub,

have you finally found the reason for the non-specific staining and a solution to it? We have kind of a similar problem. We are performing IF staining of bone marrow derived cells using a range of different antibodies. For the majority of antibodies we get non-specific staining of almost all cells. However if we use in vitro samples, we get a specific signal. And also if we use peripheral blood- instead of bone marrow- derived cells, we don`t get this background pattern of non-specific staining. So this problem must be related to the composition of the bone marrow- high fat content???

We have tried blocking, different fixation methods, permeabilization, different antibody clones etc. but this does not solve the problem.

Have you found a way to reduce the non-specific signals? Was it really due to autofluorescence?

thanks for bringing this to my attention. much appreciated.

cheers

John

Hi, it looks like you stained extracellular space.

First I would use DAPI staining to find out if the signals are not infiltrated immune cells. Without DAPI staining and information about what is stained with green is hard to tell.

Hi Mickey

poor amount of RNA you have. The best approach is the bioanalyzer, but even though you won't have enough RNA.

I can advice you to test one PCR with little amount of RNA, taking a very fresh extracted RNA and one among the RNAs you want to save.

easy, fast and low consuming in your case.

fred

Check this paper: https://www.physiology.org/doi/abs/10.1152/ajpgi.1997.273.2.G322?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub%3Dpubmed

Sometimes those ratios depend a lot on the elution solution which you used. A good old style is just to run a gel and look for smear. Do you have a bioanalyzer in your institute? Its even easier...., if they are above RIN>6 its fine for a qPCR.

We had good success, when we wrapped a small piece of adipose tissue in aluminium foil and then immersed it in N2 cooled isopentane.

Best regards, Daniela

Dealing with poor quality RNA is never ideal. If it is possible to re-run the experiments (with larger N so that your power doesn't go super low if a few samples fail), that is always the best solution. That being said, sometimes this is not possible.

What type of gene expression analysis are you trying to do? If you are doing qPCR, you can try running it on your poor quality samples and see if your reaction quality is good by looking at melt curves and the amplification of your housekeeping genes. If you want to conduct RNAseq, while poor-quality RNA is far from ideal, there are methods developed to deal with degraded RNA. Illumina's TruSeq RNA Exome reagents, for example, are design