Tissue - Science topic

For long term storage even at -20°C, I use glycerol to maintain the solubility and compactness of the protein. I get clear bands even after 6 months of storage.

You detected precipitation after storage, which I think might be resulted from the loss of proteins stability during storage. I hope it will be helpful for you.

This method typically has the soil samples collected a year (or more) after the imagery date and tissue testing. Site-specific soil test target levels are established for each field and nutrient management plans developed to reach and maintain the target levels. Plant tissue analysis gives a much more direct measure of what the plant is using; the procedures are universally applicable (in contrast to soil testing methodology); and regular plant tissue testing enables plant nutrient status to be monitored. Precision fertilization is applying the Right Input, at the Right Amount, to the Right Place, at the Right Time, and in the Right Manner and help farmers reduce the amount of inputs they use, which can help reduce the risk of environmental pollution. Precision agriculture is the science of improving crop yields and assisting management decisions using high technology sensor and analysis tools. Precision fertilization and precision irrigation are a fertilization technology based on the comprehensive analysis of the yield data of different spatial units and multilayer data, such as soil physical and chemical properties, diseases, pests, and climate. A fertilizer recommendation is the research-based set of guidelines, or management practices, for supplying fertilizer to the crop to achieve yield and quality goals (economic) in a manner that minimizes nutrient losses to the environment. High-spatial-resolution UAS imagery enables much earlier and more cost-effective detection, diagnosis, and corrective action of agricultural management problems compared to low-resolution satellite imagery. Therefore, UAS can address the needs of farmers or other users, enabling them to make better management decisions with minimal costs and environmental impact.

Hi,

I have just started to work on brain slices too. I am not that good in it but what I found our by myself and other people suggested me:

1. Different brain reagons has different protocols. For example angel of tissue cutting so protocol between different brain areas can be slightly different.

2. Cell are very sensitive for different factors, but the strongest one is mechanical stimulus, or how other would say - how you handeling, transporting your brain slices. For example if you are saturating your cells' solution with too strong flow of carbogen gas it might generate mechanical waves that over time will damage your slices. Adding to that, if your slices are big then during transportation it can bend and damage cells.

3. Sometimes cell are depolarized not because of cell quality but problem in electrophysiology setup itself or how you approach the cell. What membrane R you see after patching? Cells that you patched that are around 1-2hr after your killed mice, if you keep cell at around -70mV for 2-3min, can you evoke action potentials?

4. Speed is very important. Cell are slowly dying, so shortening recovery period is better. On other hand, you will see better which cell are still good after longer incubation.

Hi Jagtar Singh

Yes, you can use any tissue from the same group to perform IHC, IF or H&E. It is not necessary that all the staining has to be performed from one animal tissue of the group.

Hope it helps,

Thanks,

Hello Evangelia Fourli

Use proliferating cell nuclear antigen (PCNA). PCNA is present in proliferating cells during every phase of the cell cycle, peaking from G1 to S and decreasing at G2/M, On the other hand, pH3 is a marker of M-phase cells.

I suggest that you may use both PCNA and pH3 expression since PCNA expression can also be detected long after cell cycle exit and can also indicate DNA repair or cell death. If you use PCNA alone as a proliferation marker, it may overestimate the number of cells progressing through the cell cycle.

You may want to refer to the articles attached below for more information.

Best.

Thank you for the answer! I'll review my protocol again.

Isolation of intestinal epithelial cells (IECs) is one common method for studying the intestine, especially when focusing on epithelial-specific functions or interactions. However, it's not the only method available. Depending on your research goals and experimental design, there are several other approaches you can consider:

Hi Jhilik Dey

Here's how to measure mean fluorescence intensity in a few simple steps, assuming you're using Fiji (ImageJ) and analyzing a confocal microscopy image:

Hope it helps,

Thanks,

It is actually a case of freezing artifacts that often appear as Swiss cheese holes in the tissue sections. They indicate the regions where ice crystals have ruptured the tissue. Slow freezing allows water molecules to line up and form crystals, which can destroy cell membranes and create holes in loose connective tissue. Verify your flash freezing procedure and see if your fixative agent, MeOH, contains any water.

I was wondering about the thickness of your tissue sample. Considering the limited depth of the sensor to the plate bottom in the XF96, it would be much better to try it in the XF24.

Best of luck.

Hi Anna. Did you find a suitable antibody that works ? If so would you mind sharing which one ?

Thanks !

Hello Kayvan Masoudi :)

I'm sorry that you are still having issues with your extraction.

Do you use a piece of tissue or microtomy sections?

With my protocol, I cut microtomy sections of 5 microns, in order for the tissue to be thin and I don't need to use mortar and pestel. I don't use too much tissue (max 6 sections) otherwise it'll clog the columns.

If you are using too much tissue maybe it's the reason why the lysis is not complete.

I use the Qiagen DNA FFPE extraction kit

QIAamp DNA FFPE Tissue Kit | FFPE DNA Extraction| QIAGEN

Cat. No. / ID: 56404

For RT-qPCR,I would advice you to store the tissue in LN2,and then extra the RNA immediately.

Go to this site (https://xenabrowser.net/datapages/?cohort=TCGA%20Colon%20Cancer%20(COAD)&removeHub=https%3A%2F%2Fxena.treehouse.gi.ucsc.edu%3A443) and click on gene expression RNAseq -> IlluminaHiSeq* and then you can find download link.

Hi Eman Amin

Yes, you have to increase the permeabilization, blocking, antibody incubation and washing for the thick slices.

You can refer to the following protocol.

Thanks,

Yes, I added conc 2 drops:300 ul (buffer) which is even more concentrated than the manufacturer's advice.

Malcolm Nobre thank you very much for your reply! It's clear now.

Hi, Naile Merve Güven

Analysis of brain regions such as the substantia nigra, striatum, brainstem nuclei, cortex, and hippocampus is commonly conducted in rat models of PD. These areas are crucial because they show the neuroinflammatory reactions, changed neurotransmitter levels, and neuronal degeneration typical of PD. Examining these domains facilitates comprehension of the disease's dopaminergic neuron degeneration, protein aggregation, and cognitive impairments. While utilizing total brain tissue homogenate offers a more comprehensive evaluation, focusing on particular brain regions enables a more precise investigation. Ultimately, the decision is based on your study’s goals, the resources you have at your disposal, and the amount of detail needed.

Good luck!

Collagen fibers play an essential role in the activity and function of various tissues in the body. Here is an explanation of the relationship between collagen fibers and tissue activity:

In summary, collagen fibers are a fundamental component in the structure and function of tissues. They provide structural support, and elasticity, facilitate cell communication, support the wound healing process, and regulate tissue responses to stress. Therefore, the presence of healthy and intact collagen fibers enhances tissue activity and maintains their functions normally.

Yes you could combine the RNA from different columns together.

The Qiagen kit also says you should get 10-20ug of RNA from lungs, assuming 30mg. However, the binding capacity for the column is 100ug. So in theory, you could use 150-300mg tissue and still be under the max binding capacity.

Depending on how the samples were frozen years ago, flash freeze in liquid nitrogen then store at -80 vs just putting them in the -80 to freeze, could definitely contribute to degradation.

For short term storage and make sure to tightly seal them.

Yulia A. Kostyukova Thanks for your reply

Thank you very much Sebastian, interesting to know for the powder.

This is not for omics analyses but rather "simple", regular Western-Blot/RT-PCR (detection of a given transcript/protein).

I don't know if this makes a difference.

My recipe was the proposed method.

sorry

Peter

We don't know of any kits for pTDP-43 at the moment, but there are a few suppliers of ELISAs for TDP-43 which claim rat reactivity, including Abbexa, MyBiosource, and St Johns Laboratory.

Hi Omar, Thanks for explaining and suggesting the OCT removal and fixation by Paraffin.

We Paraffin embedded tissues but they were ruined, so we are trying to retrieve tissue from OCT and fix with Paraffin.

I will follow your suggestion.

The reason the Bullet Blenders are uniquely appropriate for tissue dissociation is that you can lower the speed so much. On most bead mill homogenizers, the minimum speed is relatively high, meaning that you will end up homogenizing most of your cells.

FYI, no homogenizer will reliably produce results as good as a gentleMACS when it comes to cell isolation / tissue dissociation, as the gentleMACS is specifically designed for tissue dissociation. Homogenizers will almost always have a lower yield of viable cells. The comparative upside to a Bullet Blender is the much lower price and higher throughput. If your manual method worked better than the gentleMACS and you are going to prioritize yield and viability above all else, then I would suggest you stick with your current manual method.

You can also do multiplex ELISA. You have to order a custom plate containing your markers. However it would be quite expensive

Hello, You don't necessarily need to pulverize your samples before extraction with Trizol; I have seen this procedure being used more with plants. For animal tissue, you usually add Trizol directly onto the tissue while it's still frozen, or just thawed (preferably still frozen).

In our laboratory, we usually aliquot enough tissue mass for extraction (between 50 and 100 mg) into a new tube free of DNAses and RNAses, and immediately add 1 mL of Trizol. This sample is then homogenized using the BioSpec Tissue Tearor (we've tried other brands and never got good results) or a pestle. To ensure that you preserve RNA, it's important to follow the protocol steps rigorously, minimizing any contamination by endonucleases as much as possible. Use gloves and reagents suitable for molecular biology and good laboratory practices. With mouse tissue, you probably won't have any problems. The link recommended by Uma Dharshini K Vijayamuthuramalingam is very worth reading. You should also read the complete Trizol protocol, Troubleshooting, and FAQs.

Sincerely,

Your problem is going to be that nothing other than a barn-door difference is going to be significant. If you really want to distinguish between cancer and polyp tissue, this is too important a question to be decided on the basis of small numbers. The only thing that a small sample is useful for in a case like this is where there is a stunning difference between the groups – passes the Mark I Eyeball Test – indicating that the marker has possibly got clinical potential.

Firstly, full disclosure - I make vibratomes!

Assuming you are following a standard protocol for thawing, then 50µm sections from fixed tissue should be easily achievable with a reasonable vibratome. Agarose embedding can help with curling prevention, but should not be necessary.

I would certainly reduce the speed as that is quite high, and reducing amplitude should help with delicate samples. What blade frequency are you using?

However, I would also look at the blade you are using. Don't let anyone convince you that a standard razor blade is as good as a purpose made stainless steel blade or ceramic blade. Razor blades are relatively blunt, designed to cut hair and not skin, and introduce compression (I am not saying this for any commercial reason, it is fact!).

If you have the opportunity to use fixed tissue that won't have had the cryostat treatment, you could ascertain whether the freezing and thawing regime is the problem.

I can see your question is a few months old, but hopefully I can help. (Full Disclosure: my Company makes vibratomes!).

As one answer states you have several variables to play with (dependent upon the model of vibratome):

Whatever the tissue and application, lower frequencies and lower amplitudes inevitably mean lower friction and shear stresses in the tissue.

As a general rule:- tougher tissues, for example aged brain, cauterized brain, diseased human heart and rodent heart will require wider amplitudes and/or higher frequencies to cut them, whereas if tissue are more delicate and/or easier to cut they can be preserved better with lower frequencies and smaller amplitudes.

To answer your specific question technically, if you increase the blade amplitude (i.e. the blade has to travel further in one cycle) to maintain the blade frequency the blade travels at a faster speed. This can be useful to cut tough tissue as mentioned above.

This paper and/or authors may be able to provide further insight:

Z-axis deflection could also be relevant for your application. Your particular Leica model does not have the option to measure this and, therefore, to correct for it unfortunately (the higher Leica version does). Every time you change a blade you introduce a Z-axis deflection as you can never position the blade in the exact plane of the horizontal movement. It is necessary to measure this deflection to then correct for it (Campden and Leica offer a device to achieve this). You can get an overview of this from this article:

When it comes to compression, compression is primarily introduced by poor blade angle and poor quality of blades (e.g. razor blades - designed to cut hair not skin!). The blades we use are bevelled to a specific angle to complement the blade holder angle such that you shave sections off the mounted specimen and do not introduce any horizontal or downward compression.

I hope that helps.

i would always blank with whatever i have eluted my RNA with my i use silica columns but i guess you could do the same principle. blank with qiazol then measure your sample just a suggestion. But from the curve there may be contaminants at very close wavelengths.

We typically employ the TRIZOL method for extracting total RNA from tissues. I am unsure if the TRIZOL method can be used in this case, but I believe it is worth attempting if the dye does not interfere with the TRIZOL reagent and does not degrade the RNA.

Betalains are the pigments responsible for the characteristic red colour. It is a water soluble pigment. This pigment definitely interfere in the extraction of DNA. This can be easily degraded by heat, light and oxygen. The tissues of cacti can be exposed to deionized water at above 60 degree centigrade at different time intervals (temperature and time can be optimized to get rid of the pigment) . Then, the tissues can be used for DNA extraction. This will to a maximum extent remove the pigments. Better use the CTAB method for extraction of DNA.

Hi Francisco,

I am also curious if you succeed with RNA isolation. I am having the same problem; my pellet is very viscous, and I am looking for a solution right now.

Thanks,

Tetiana

Hi,

liver is just one of those tissues that is known to high a very high autofluorescence. I would suggest staining your markers with a PE or even preferably APC fluorochrome as then you can simply filter out the signal in FITC as autofluorescence.

I also worked with LC3-II once, in highly AF tissue and I quenched this by using an incubation with Sudan black. Maybe read this attachment.

Best of luck!

Cristina

Hi Aamna Kausar

Yes, Tunnel assay can be performed in formalin fixed tissues. Several commercial kits detect apoptosis in formalin fixed tissues. Please find the link to kits.

1. https://www.thermofisher.com/in/en/home/technical-resources/research-tools/image-gallery/image-gallery-detail.22089.html

2. https://www.promega.in/products/cell-health-assays/apoptosis-assays/deadend-fluorometric-tunel-system/?catNum=G3250

Thanks,

I think this is the key:

"I want to compare the two sets of data to look for any differences in level of protein in the two different brain locations over the 4 time points."

From each group of 5 mice you measure the protein concentration in two different brain locations. This is where you get the "two sets of data". And, yes, if you wish to say so, these data are paired between set 1 and set 2, because these data are from the same mice.

But when your data is complete (no missing values) it would be the simplest solution to calculate the ratio of the concentrations in the two locations for each individual mouse. So you end up with a single "set" of data, 20 concentration ratios for the 20 mice in total.

These 20 ratios are organized in 4 groups (each with 5 values from one time-point). All these ratios are statistcally independent, if there is nothing in the experiment that could serve as a common source of variance*, and can be analysed as such. These ratios are not normal distributed, so if you like to get p-values make sure to use the logarithms (log-ratios).**

* In the worst case, all mice in one group were kept in one cage, so that cage and group are fully confounded. In such a case there is no independent value in any group and you would not be able to answer your research question, because any difference you see in any particular time-point could be due to something related with that particular cage rather than with that time-point.

** Another way is to start with log-concentrations and calculate the differences of the log-conc between the two locations in each mouse. Yet another option would be to use generalized linear model of the quasi-Poisson family, but this is rarely used.

There will likely be significant batch effects. I would analyze each set separately to get a higher power (which will be the case when the variability between the sets is large and won't be compensated by the reduction of standard errors due to the larger sample size).

You might consider pooling the p-values according to Fisher's method, if you need a single p-value per gene.

I'm looking for the same type of answers... did it work snap freezing the tissue in liquid N2? I would like to do it in E15 and E17 mouse cortex samples. TIA!

Hi Cesar,

Have you validated these cells with neural markers to rule out the possibility of cross-contamination from other cell types? It can often happen during the isolation procedure. At the onset of the pictures, it looks like stronger cells (such as cancer) trying to overtake the primary neuronal cells.

Vortexing is safe, less prone to contamination and easy to perform. However, vortexing may not be sufficient to homogenize some types of tissues, such as collagen-rich or elastic tissues, that require more mechanical force to break down . Vortexing may also cause heat generation and foaming, which can affect the quality and yield of RNA .

Therefore, it is recommended to use vortexing in combination with other methods, such as freezing and thawing, sonication, or enzymatic digestion, to improve the homogenization efficiency and RNA integrity. It is also important to optimize the vortexing time, speed, and temperature, and use appropriate buffers and stabilizers to protect the RNA from degradation .

If you want to learn more about vortex to homogenize tissue, you can check out these web search results:

Enhancing histological sectioning techniques to improve the quality of tissue requires attention to various factors involved in the process. Here are some strategies to enhance histological sectioning techniques:

1. **Fixation and Processing:**

- Ensure proper tissue fixation using appropriate fixatives to preserve cellular structures.

- Optimize tissue processing techniques to maintain tissue integrity and prevent artifacts.

2. **Embedding:**

- Use high-quality embedding media to support tissue during sectioning.

- Embed tissues uniformly to avoid uneven sectioning.

3. **Section Thickness:**

- Optimize section thickness based on the type of tissue and the analysis requirements.

- For certain applications, like immunohistochemistry, thinner sections may be preferred to enhance antibody penetration.

4. **Temperature Control:**

- Maintain consistent temperature conditions during sectioning to minimize variability and improve reproducibility.

Continuous improvement in histological sectioning techniques involves a combination of technical expertise, equipment optimization, and adherence to best practices. Regularly reviewing and updating protocols based on new advancements in the field can contribute to enhancing the overall quality of tissue sections.

This works for well-attached sections.

Too vague, what kind of drugs? I would use Google it first to see if it was done before.

Your problem is the speed of sectioning. When you take a look under the microsscope you will see that your sections partially distroyed. They show a fiber like appearence. Rolling and the fiber like section-structure show you that the speed is too high. Slow down and play with the temperature of your cryostate, Mount some of your sections directly on a slide and check the result under the microscope

Terika Smith Did you manage to this successfully? I'm thinking about doing the same?

You are describing non-specific cytoplasmic binding. I'd consider either IF in FS or chromogenic IHC (peroxidase, phosphatase) in FFPE

Fritz Caspers

You may already know this!

rfid in this band uses a very weakly coupled resonant air-cored magnetic transformer. Read the application notes - they are interesting and fun. It isn't an antenna. It is in the inductive near field, where the coupling is 1/r³. The tag is the secondary and receives enough power to turn on, and modulate its own impedance, which is detected as a very small impedance change in the primary. The system doesn't radiate (much), so it is like a toothbrush charger, but much more weakly coupled. Power goes into the tag, and is wasted only to losses in the primary, and to eddy currents in any surrounding metalwork.

First, step of thawing the slide is very critical. Place slide flat horizontal, not in coplin jar, let the OCT melt for 5- 10 minutes. Than wash the sections by pouring PBS around section. Idea is to remove OCT from under the section. If there is thin layer of OCT left between section and slide, then section tend to slip. Do 4 washings very gently, so that Whole OCT under the section is removed. Then section will settle down and will stick to the slide. Also use slides which are suitable for cryo. Now a days, good quality slides are available from many compnies. Be patient at this first step. Generally we ignore first step. Keep slides flat always.

Dear Anna,

I am also worry about removing of OCT can be affected to RNAseq quality. Have you tried to remove OCT and proceeded the RNA seq?

To me this doesn't quite look like smearing of a stain. The edges are too sharp, the structure is too well defined. A smear rarely has this kind of structural integrity to it. You've mounted it in ProDiamond with DAPI though. What this means is that there is always DAPI around preferentially accumulating in regions it has high affinity for. It will also stain RNA, although in, for example, a cell grown on a coverslip the intensity of this fluorescence is so much lower than that from the nucleus that you never really see it.

The slides are also quite old. I know you've said that they're stored at -80C, but this is just what you've been told. Perhaps a collaborator left them out on the bench over the weekend once, they may have a -80C freezer which really should be replaced as it isn't uniformly keeping everything at that temperature. The slides have also been shipped around. There is a possibility that there is some sort of bacterial/fungal growth in those regions but at that resolution you wouldn't be able to tell.

I'd suggest doing two things. The first is zooming into a section where that streaking is quite pronounced and taking the highest resolution image you possibly can in order to see if the stain is uniform or punctate. This might hint at some sort of contamination.

The second is to ditch the ProDiamond with DAPI either get the pure chemical and make up the stock yourself or buy one that is preprepaired. If you see papers where people have used, for instance, a 1/10,000 dilution of their stock and stained for 10 minutes. Dilute the DAPI at say 1/100,000 and add it along with the primary antibody. With all the subsequent washes and incubations you will thoroughly rid the tissue of any DAPI that's not strongly bound to something. You should end up with pretty close to zero background and intensly bright staining everywhere you expect (ie the nucleus).

If the intensity of the streaks is still as bright as the nuclei under these conditions it would be hard to argue that it's binding to anything other than DNA which for whatever reason has ended up in those locations. If the difference in intensity between the nuclei and the streaks has greatly increased perhaps its due to huge amounts of RNA in those areas.

You won't get a conclusive answer with one or two slides but it may help point you in the right direction.

Frozen sections are usually performed at -20 degree Celsius. It is possible that at lower temperature you experience separation between the section and the medium. You might also try the old trick to warm up the surface with your thumb before cutting. Each tissue has a slightly different optimal temperature for cutting.

Best,

Giorgio

Suggest you test it.

Hi Gurjit Singh ,

ORO can only be used for frozen sections (cryosectioned).

To use a bead beater to isolate live bacteria from mouse tissues, you will need the following materials:

Once the culture plate has been incubated, you can count the colony forming units (CFUs) to determine the number of live bacteria in the mouse tissue.

I can give you my opinion, although I am not an expert in the field. It can't be both slimy and powder... If its slimy my best guess is DNA so try to increase the DNase. If its powder, perhaps its calcium salts, so you can increase the EDTA or add a salt- cell cleaning solution. Do you have o picture of the precipitate?

Hello Serena, from which species is your tissue? Is it rat tissues which you like to investigate? The reasonis is your primary antibody because when you detect the primary ab out of rat and you use an anti -rat secondary you stain all the serum components of the rat section. That's why your negative control looks like your experimental section. I recommand in that case, use a primary antibody from any other species.

If you are working with mouse tissue you need a mouse absorbed secondary antibody.

The 3rd rason could be, indeed, the blocking step. Here you have to test systematically which part of your blocking and dilution medium are responsible for this result. On recommandtion: if you use BSA or fat dry milk, replace it with normal serum (blocking step 10%, dilution medium 1%). Use the normal serum from that species in which your secondary ab is generated, in your case from Goat. Good luck!

Thank you.

I am looking essentially for dissociation condition tips.

I have already improved different steps of the fixation/dissociation protocol (mincing tissue into 1-mm pieces, increasing digestion time, testing different enzymes, adding a mechanical dissociation step), and still doing in the pilots. But I was wondering if anyone has a good dissociation enzyme that dissociate small skin pieces and keep a good cell integrity.

On Which tissue you are working? first check wether your antibody is good or not, (by taking cell lysate that has your protein of intrest from cellilne) did you ensure transfer of proteins on membrane after blotting (By ponceau staining)

Prepare your tissue lysate by homogenization in RIPA buffer (50 mM Tris-HCl, pH 7.4 + 1% Triton X-100 + 0.2% sodium deoxycholate + 0.2% sodium dodecylsulfate (SDS) + 1 mM sodium ethylenediaminetetraacetate + 1 mM phenylmethylsulfonyl fluoride + 5 µg/ml of aprotinin + 5 µg/ml of leupeptin). The lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl, pH 6.8 + 12.5% glycerol + 1% SDS + 0.01% bromophenol blue) containing 5% β-mercaptoethanol. (Check whether your antibody will work in reduced condition from the antibody instruction manual).

Best Wishes

Hi Eef Dirksen,

Thanks for your comment.

I have some serum and tissue samples from patients with a certain disease, and I want to identify signature proteins or peptides that can differentiate them from healthy controls. I am wondering what is the best method to extract and analyze (top down or bottom up) these biomarkers.We have a Q-Exactive mass spectrometer for this experiment. how to handle the data analysis and interpretation? Is there any specific software or tools to process and visualize the results? 

Generally supernatant and sediment are separated by centrifuging or filtration.

Parafin embedded tissues can be used for immunofluorescence staining. However, like fresh frozen tissues, there are caveats, specifically your antigen of interest, optimizations by the antibody manufacturer, and your own optimizations for your particular tissue/antigen. Most manufacturers will list and reference whether or not a particular antibody has been tested in paraffin embedded tissue. Additionally, optimization for immunofluorescence markers is more challenging to produce good quality images, with specific positive staining and reduce background/ non-specific stain. Finally, paraffin tissues are usually cut very thin 5-7uM, as such your depth of field is limited.

More information is needed to fully address this question. One additional point, if your question is that of "can you use the same sample and concurrently stain H&E and Immunofluorescence simultaneously..." that would not be recommended.

Did you treat your samples with DNase I? This might be gDNA contamination.

Hello Kartarzyna, it depends on the kind of your tissue. If you are work with compact tissue like brain, liver, kidney, spleen you can treat 20 to 50µm wtih a immunhistochmical protocol. Even here it depends on the protein you like to detect. The free floating method is very commen in neuroscience.

Here a short protocol:

use microtiter plates with 24 wells; each row of the plate is one working step.

1. Rinsing 3 x 10 min in PBS/TBS

2.Blockin of endogenous peroxidase (ony with Peroxidase containing detection systems).

3. Rinsing 3 x 10 min in PBS/TBS

4. Blocking of unspecific antibody binding (with normal serum or BSA) -- 90 min

5. Primary antibody (dilution has to be testet at first) -- overnight

6. Rinsing 3 x10 min PBS/TBS

7. secondary antibody biotiniylated or with Peroxidase or with fluorophores labeled -- 90 min

8.Rinsing 3 x10 min PBS/TBS

9.. StreptAvidin peroxidase or Fluorophore labeled -- 90 min

10. Rinsing 3 x10 min PBS/TBS

12. DAB-Staining for brightfield analysis 5-10 min

13. Rinsing 3 x10 min PBS/TBS

14. mounting on gelatine coated slides

13 dehydrating, clearing and mounting in a xylene soluble mounting medium (DAB)

or mounting with a water solubel mounting medium with or without Dapi for immunfluorescence

your dilution medium should contain 02.% Triton-X 100 and 1 % normal serum

The reaction should be processed on a rocking-table or shaker at room

temperatur. You can put your plate in the fridge but depending on the antibodies you will need higher concentrations.

Every antibody concentration has to be tested before use.

Good luck!

In normal tissue, a biomarker is a measurable characteristic of a biological system that is indicative of normal function or its response to an external factor like therapeutic intervention.

For instance, in radiotherapy, one could look into functional assays such as DNA double strand break repair, induction of chromosomal aberrations, or radiation- induced apoptosis in exvivo irradiated blood lymphocytes that could be used as predictors of radiosensitivity. Also, a number of IR-induced transcriptional and translational alterations could be studied. The use of cell lines for predicting biomarkers in normal tissue is still a controversy.

You may want to refer to the articles attached below for more information.

Clin.Oncol. 2013,25, 610-616

Mutation.Res. 2017, 771, 59-84

Best.

Yes, it is possible to process tissue for FFPE after cryoprotection in sucrose, with some additional steps:

- Wash the sucrose-cryoprotected tissue thoroughly in several changes of PBS to remove all traces of sucrose. Sucrose can interfere with tissue processing and embedding if not removed.

- Post-fix the tissue in 10% neutral buffered formalin for at least 24 hours. This ensures adequate cross-linking and preservtion for FFPE.

- Process the fixed tissue using standard FFPE processing protocols - dehydration through graded alcohol and xylene, infiltration with molten paraffin wax, and embedding into wax blocks.

- The initial PFA fixation allows switching methods, but post-fixing in formalin ensures optimal tissue preservation and cross-linking for FFPE sectioning.

- Take care to orient the tissue properly during embedding into the wax block.

We need canals instead of using tubes. The curved canals and stoppers could make changes on waters regime to decrease the speed of the water from topsides to the downsides. In Iranian ancient methods, we used the an ancient canals under the ground that was called Qanat. This means, changing directions with another smaller canals could reduce the speed and the water flows from topside to the downsides

I am not really familiar with phenol extraction, but phenol itself is moderately soluble in water (8:100) so I think you can just wash it over water several times?