Neurobiology and Brain Physiology

Neurobiology and Brain Physiology

  • Thomas E Salt added an answer:
    How can I get stable and perdurable LTD in hippocampal slices from adult mice?

    Hello everybody.

    I have seen through publications that some labs have been routinaly able to induce very long-lasting in vitro LTD in young and adult animals. I would like to ask you if you can give me some details of the methods to achieve it.

    I am trying to get LTD in my hippocampal slices from ~10 weeks old mice, but so far it has been almost impossible. I have been using both electrical stimulation (single 900 pulses at 1 Hz) or the application of mGluR2/3 agonist LY354740. I am stimulating the medial perforant path and recording fEPSPs in the dentate gyrus, at room temperature.

    Does anybody have some advice to get stable LTD?

    Thank you very much in advance for your contributions.
    Diego Fernández

    Thomas E Salt · University College London

    Hi Diego: I agree with the previous comments... but slice health (protection while cutting) and then temperature during storage and recording are very important.  Recording at 'room temperature' is probably not ideal - I would suggest 32-33C.

  • Pengfei Liang asked a question:
    Does anyone know how long can squid giant axon survive in sea water for Na/K pump study?

    we are trying to study Na/K pump on squid giant axon. But can not fish one in florida during summer time. So we are trying to get one from Woods Hole Marine lab. Just wonder how long it can survive in the sea water via shipping?

    Thank you

  • Mehdi Kargar added an answer:
    What are the neuroscience correlates and the difference between a thought and spoken sentence?

    Speech is a complex phenomenon requiring motor system. Motor system learns from the feedback received from proprioceptive and other sensory systems and involves an intricate play between basal ganglia and cerebellum along with motor cortices. But how are thoughts formed and how regulated ( don't know whether we can call it regulation but appropriate formation). It is definitive that thoughts which can be communicated require language (and hence speech comes into picture). But how exactly the neuroscienctific mechanisms differ in the two cases is my question.

    Mehdi Kargar · Khorasan Institute of Higher Education

    hi Dear patil

    plz see this link :

  • John Kudolo added an answer:
    Do you know any direct efferent projections to cerebral cortex from pontine nuclei other than cerebellar cortex?

    The neurons in the pontine nuclei have the axons which project to the purkinje cells. But I don't know whether the pontine nuclei have the projections direct to CEREBRAL cortex or not. Does anyone have data or suggestion?

    John Kudolo · Cytocentrics GmbH

    Hi Yoshiyuki,

    There is a projection from basilar pontine nucleus neurons projecting to the grnular cells via the mossy fibers and then unto the Purkinjie cells in the cerebral cortex. There is also a direction projection from the basilar pontine nucleus to the deep cerebellar nuclei base on anatomical studies in literature. I have incomplete and unpublished optogenetics data on basilar potine nucleus neurons and it projection to the granular cells in the cerebral cortex

    I hope this Information is helpful to you, thanks 

  • Enrico Marani added an answer:
    What is the best way to enzymatically dissociate mouse brain tissue that gives a high yield for all cell types (neurons, astrocytes, microglia)?

    We currently use mechanical dissociation, but our astrocyte yield is low. We've tried papain (I'm not sure which concentration we used), but it did not give a good yield for neurons. We want to minimize cell death during dissociation as well as maximize preservation of cell surface markers (e.g. CD11b). Any suggestions? Thanks!

    Enrico Marani · Universiteit Twente

    Mechanical issociation is the best method.It is your culture medium for dissociation that determines the yield.

  • Deforest Mellon added an answer:
    What is the best method of DiI delivery for fixed cerebellar tissue?


    I am using lipophilic dye to label Purkinje cells in coronal fixed tissue. From the variety of options (Paste, Dye Crystals, Pellets), which one will provide the best result? I am also planning on doing IHC with the same tissue, so permeabilizing reagents will play a factor. 

    Thank you,


    Deforest Mellon · University of Virginia

    We have had good luck using DiI and DiO by making a paste of the crystals with vaseline. The DiI crystals dissolve in the vaseine and it can then be applied to nerve tracts using a tiny insect pin under the microscope for application.

  • Matija Fenrich added an answer:
    Any suggestions on the roles of hexokinase II ?

    Hexokinase I is predominant form of hexokinase family in mammalian brain. But under hypoxic conditions, there is no obvious change in its expression. In contrast, hexokinase II, which is mainly expressed in insulin-sensitive tissues and malignant tumors, experiences a robust upregulation. I think it is a norm that only when a protein's function has been compromised,  then  its subsitute may undergo a change to adjust to this situation.So my question is why hypoxia exposure can induce a significant uptegulation of hexokinase II whereas it has no effect on hexokinase I in brain? Is it possible that it may play some other roles in hypoxic situations? Thanks ~~

    Matija Fenrich · University of Osijek

    Dear Yuan Lee,
    Hexokinase II binds to the mitochondrial membrane through interaction with the outer membrane voltage-dependent anion channel (VDAC), preferentially at contact sites between the outer and inner mitochondrial membrane. This location is important for the integration of glycolysis with mitochondrial energy metabolism, since VDAC interaction with Bcl-2 antiapoptotic proteins controls the rate of release of mitochondrial intermembrane space proteins that activate the execution phase of apoptosis.
    Therefore, Hexokinase II binding to VDAC suppresses the mitochondrial permeability transition and inhibits apoptosis, thereby contributing to the neuronal survival of hypoxia. That is why hypoxic conditions upregulate expression of this enzyme.
    On the other side, Hexokinase I has no effect on Bcl-2 and apoptosis regulation, which makes it not useful in survival of hypoxic cellular stress.
    I hope this helps you.

    For more info see:

    Chiara, Federica, et al. "Hexokinase II detachment from mitochondria triggers apoptosis through the permeability transition pore independent of voltage-dependent anion channels." PloS one 3.3 (2008): e1852.

    Pastorino, John G., and Jan B. Hoek. "Hexokinase II: the integration of energy metabolism and control of apoptosis." Current medicinal chemistry 10.16 (2003): 1535-1551.

  • Wasiu Gbolahan Balogun added an answer:
    I need information on air freshener substances and their effects on the brain

    i am looking for research materials on the effects of air freshener substances on the brain, i have seen some materials but they are not scientifically backed. i know some of the substances causes toxic encephalopathy, dementia and other neurological diseases but i need their scientific basis

    Wasiu Gbolahan Balogun · University of Ilorin

    Thank you Helena Barros, i am grateful for the explanation. I am looking for literature's on air freshener substances like p-dichlorobenzene and their effects on the brain either histological or biochemical. i will be grateful if you can share some of your materials with me. my email address is Thank you Janet Mcalister, i am grateful for the paper but i will be happy if there are other chemicals you can share with me

  • John S. Antrobus added an answer:
    How Are Memories Retrieved in the Brain?

     Greg Miller " Many details of the process of memory recall are not known (or are disputed). Even so, some researchers say it's time to revise some aspects of the standard view—such as the notion that the hippocampus is not involved in retrieving older episodic memories, and that memories become fixed and unchangeable once transferred to the neocortex. Newer work suggests a far more fluid role of memory, and one in which retrieval plays a crucial role in shaping memory over time"

    John S. Antrobus · CUNY Graduate Center

    You might like to look at O'Reilly's emergent neural network models .There are many kind of priming - all work somewhat differently.

  • Alejandra González-González added an answer:
    Can someone recommend a neuronal tracer that diffuses in short time in fixed tissue?

    I used DiI and I traced very well some fibers, but a disadvantage is that when it's exposed to UV light in a microscope, loses fluorescence very quickly. I need a tracer in fixed tissue because it's easier to administrate the tracer in the zone that I'm studying than in stereotaxic. Thanks.

    Alejandra González-González · Universidad Nacional Autónoma de México

    Thank you very much everyone, especially Justin, valuable information, now I'm performing my experiments and I am incorporating your advice.

  • Ravinder Jerath added an answer:
    Have Mohamed Koubeissi's results regarding the ON/OFF switch of consciousness been successfully reproduced?

    (And how difficult would it have been for the researchers to resist the urge to zap their poor subject in and out of consciousness at will?)

    Ravinder Jerath · Augusta Women's Center

    Adam, I read information regarding Kubeissi's  findings with his patient as noted below. 

    An accidental discovery, this effect was uncovered during investigations into seizures. A 54-year-old woman fell into unconsciousness when a small electrical impulse was applied to the tiny, sheet-like claustrum. The patient exhibited no response to external stimuli during the time she was unconscious, and her breathing slowed significantly.

    Please note it is stated here Kubesissi also found that the breathing slowed significantly. my paper on sleep hypothesis it clear that  breathing is the rhythm that changes all body rhythms including effect upon hypothalamus and cardiac rhythm. (Central pattern ) This affect actually leads to daily turning on of the sleep switch in humens. This happens when the breathing slows down and changes the tonic firing of Thalamus to burst firing. The thalamus is effectively cut off from cortex leading us to sleep. It is possible the claustrum of this patient triggered the slow breathing leading to thalamic rhythm change. Below is my article. I also have written several original articles on consciousness. Interesting question !

  • Enrico Marani added an answer:
    Any suggestions on chromatin during brain development?

    Hi all,

    this is another of my mysterious questions. I am working with brain chromatin during development, in particular with P4 and P60 mouse brains, and I am always getting a weird result. whether I am preparing chromatin for ChIP or just isolating it after Mnase digestion, I always get a huge amount of DNA from P4 brains but very small amount from P30. Before you ask I treat every sample with almost 200ug of PK for 2 hours. My first thought was that DNA could get stuck to proteins and so I could lose it during my phenol chloroform extraction. I always start from same amount of nuclei so DNA amount should be same. Anybody can help me solve this?

    Enrico Marani · Universiteit Twente

    Think of pruning and apoptosis afterwards to non-pruned contacts.

  • Katherine Poinsatte added an answer:
    How do I reduce vessel staining after BDA injection?

    I am iontophoretically injecting BDA (biotinylated dextran amine) into the motor cortex for anterograde axonal tracing. My BDA is tagged with Alexa594. I'm doing 5 minute injections with a current of 5 uA, alternating 7 seconds on and 7 seconds off with pulled glass pipettes of 20-50 um. I'm having really nice staining of the axons but the BDA also stains the vessels near my injection site. Is there any way to avoid this or is this just an inevitable issue with the method? 

    I've attached a picture. Has anyone encountered this before?

    Katherine Poinsatte · University of Texas Southwestern Medical Center

    I don't see any lingering blood in my vessels when I'm sectioning and my brains are appropriately bleached so I'm unsure if it could be a perfusion problem. Thanks for the suggestion though!

  • Mohamed Najimi added an answer:
    When analyzing images of fibers in coronally-sectioned mouse brain, is it possible to differentiate between fibers of passage and terminal fibers?

    Does anyone have a method to determine the difference by morphology alone i.e. without the use of an additional label such as synapsin for terminals or tau for axons?  Our neurons are filled with virally-expressed eYFP.

    Mohamed Najimi · Université Sultan Moulay Slimane

    By classical IHC (using peroxidase for example), you can distinguish between fibers of passage and terminals. These latter are generally presented as varicosities, but this does not mean necessary that they correspond systematically to terminals. Indeed, depending on sectioning plans. Fibers of passage are presented as continued filaments with different sizes. In summary, you don't need specific method nor specific antibodies to visualize them.

  • David Skaer added an answer:
    What is the relationship between hyperprolactemia and mental illness?

    i know that some antipsychotic medications can cause hyperprolactemia as a side effect, but I am wondering whether the dopamine and GABA imbalance produced by hyperprolactemia resulting say from a pituitary adenoma can cause serious mental impairment.

    David Skaer · Saint Leo University

    I've had patients who were hyper after having a baby--still producing milk after 3 years (and never nursed). She thought it was normal. Doc gave her meds to dry her up, and her depression lifted. Had a few cases of depression with hyper.

    Also, had some OCD connected with hyper as well. I only have a few journal articles regarding'll have to search a bit for them.


  • Nathan Ahlgrim added an answer:
    How can I label brain hemispheres for free-floating histology?

    I am running immunohistochemistry on rat brain tissue, and the sections will be stained while free-floating.  However, we need to reliably know the left from right hemisphere.  We cannot take a notch out of the cortex (which we have done previously), because we are taking a full survey of the brain.

    Could someone recommend a method to label the hemispheres?  Is there some sort of marker or pen that is visible to the eye but would not affect staining?

    - Thank you -

    Nathan Ahlgrim · Emory University

    Thank you all for your advice.

  • Siamak Gholamalipour added an answer:
    what kind of adenosine receptor (A2A , A1) is activated in responding to the secretion of ATP by astrocyte?
    ATP released from astrocytes is degraded to adenosine and activates presynaptic adenosine A2 or A1 receptors that leads to an increase or decrease in its release probability (Panatier et al. , 2011). Now the problem is:
    After secretion of ATP by astrocyte:
    Which mechanism is activated A2A receptor on presynaptic neuron?
    Which mechanism is activated A1 receptor on presynaptic neuron?
    Which mechanism determines that what kind of adenosine receptors on the presynaptic neuron (A2A , A1) should be activated in response to astrocyte adenosine secretion?
    Siamak Gholamalipour · Urmia University

    Hi Hossein 

    please check this article 

    I hope it's helpful .

  • Katherine Poinsatte added an answer:
    How do I reduce BDA from staining the pia?

    I'm currently doing BDA (biotinylated dextran amine) injections into the motor cortex for anterograde axonal tracing. My BDA is tagged with Alexa 488 and Alexa 564. The biggest issue I'm having at the moment is that my BDA is staining the pia mater of my brain and appears to be going everywhere! I think it's contributing to some of the vessel staining I'm seeing as well. 

    I've tried varying speeds and amounts of injected BDA. Pulling the needle out slowly, but none of this seems to be really reducing the spillage. 

    I've attached a picture so you can see the staining all along the edge of the brain. 

    Has anyone who has done this procedure before encountered this problem?

    Katherine Poinsatte · University of Texas Southwestern Medical Center

    I've switched over to iontophoresis but I'm still seeing vessel staining. Sorry that the picture is a little dark. 

    I'm doing 5 minute injections with a current of 5 uA, alternating 7 seconds on and 7 seconds off with pulled glass pipettes of 20-50 um. My perfusions are good with no remaining blood in the vein. I reverse the polarity of the current when I insert my needle into the brain. 

    I'm really unsure of what I'm doing wrong. 

  • Espen Sjoberg added an answer:
    Are our animal models and the human subjects we choose still valid?
    As someone who works in a lab where behaviour of rodents is studied, I constanly ask myself the question if all the conclusions we are drawing from our experiments are the exception or the rule. I mean, we (scientists in general) use a very specific species, and a lot of the times (for instance, research with geneticaly engineered models) inbreed lineages, what drops the variability between individuals even further; or in the case of basic human research, subjects generally come from a very limited pool, either age wise or education level and etc... (Attached a publication that tackles this idea in the immunology field).
    I wonder if studying these models isn't leading scientists to wrong paths and hampering science rather than helping understanding through simplified models. Also could we just be doing science like this due to an inheritance issue? As in we inherited this paradigm of simplifying models because the available tools to analyse data weren't sufficient for biological complexity? Couldn't we with the great technological advancements now deal with the complexity and variability that will emerge when experimenting with more complex models and subject groups?
    Espen Sjoberg · Oslo and Akershus University College of Applied Sciences

    In my opinion the most important thing in animal research is the validity of the model you are investigating. In my experience some researchers tend to show a logical fallacy, namely argument from analogy: just because group 1 (e.g. mice) and group 2 (e.g. humans) are similar in one respect does not mean that new knowledge gained from doing research on group 1 will automatically transfer to group 2.

    In the medicinal sciences this validity is often justified by some physiological component, such as the common existence of a neurotransmitter or neurological deficit. In the behavioral sciences this is trickier, because we look at similarities in behavior, but these similarities can occur by chance or may be misinterpreted.

    I believe animal research is valuable and can significantly advance human knowledge and treatment, but whether it is absolutely necessary or not largely depends on each individual field, and how valid each model is.

  • Richard Godinez Tello added an answer:
    Is it known, where the harmonics in SSVEP recordings come from?
    If I use a 40 Hz stimuli, there are also peaks in spectral data at 5, 10, 20 and 30 Hz as a response from the brain. Are these generated by the brain? How? Is there a VEP, when a light switches off?
    Richard Godinez Tello · Universidade Federal do Espírito Santo

    It is usually called as fundamental frequency to f_i and harmonic frequencies as its multiples, i.e. 2x f_i, 3x f_i, ... etc. Frequency ranges are also called bands, it is known that the low frequencies have the largest amplitudes of SSVEPs. However, it is tiring to the user. On the other hand, the middle band combines user comfort and an acceptable level of SSVEP amplitude. 40 Hz is considered as High Frequency in this case. Brain mechanisms generate these potentials and their harmonics are present. For this reason, feature extractors simulate these brain processes achieving good accuracy rates.

  • Snigdha Tiash added an answer:
    Is there any in-vitro system (commercially available) that mimics blood brain barrier?

    I am trying to design carbonate apatite nanoparticles that might cross the endothelial layer of brain capillary (blood brain barrier-BBB). Thus, an in vitro system would be a better option (easy to understand and faster) to check the permeability of particles before moving to an in vivo model. I am looking for an in vitro BBB system that can be used for this purpose. I have seen the co-culturing systems discussed by many researchers. Are the co-culturing system with the filter available commercially?

    Snigdha Tiash · Monash University (Australia)

    Thank you so much for all of your time. They are really helpful

  • Yuan Lee added an answer:
    Does anyone have good rat cortex neuronal culture protocol?

    Dear all,

    We are trying to obtain rat cortex neuronal (pyramidal) cell culture with no success so far. We currently use P1-3 rat pups and procedure is as follows:

    Decapitation ->

    Decapitated head immediately placed into 96% EtOH @RT for ~5sec. ->

    Then head is immediately transferred to ice cold dissection media (PBS with glucose and pen/strept) and brain is removed (procedure takes 2-3 min)->

    Brain then placed to fresh ice cold dissection media and cerebellum, olfactory bulbs, meninges and etc. are removed this takes ~8 min. (per brain). ->

    Cortex then is transferred to the fresh ice cold dissection media and is finely (~1x1 mm) chopped with the scalpel blade (takes ~2 min) ->

    Pieces of cortex then collected and transferred to the preheated (37 C) Versene media, gently mixed and incubated 5 min in 37 C gently mixing the solution every 1 min. -> 

    After Incubation with Versene 2 different diameter fire polished Pasteur pipets are used for trituration still in 5 mL of Versene solution (this procedure additionally takes ~5 min.) ->

    After trituration 5 mL of ice cold growth media (Neurobasal-A supplemented with B27, L-Glutamine, FBS and Pen/Strept) is added to the cell suspension and mixed to deactivate Versene. ->

    To wash cells of Versene, suspension is then centrifuged 250 x g @4C and supernatant then removed. ->

    Cells then are resuspended in 5 mL of ice cold growth media and transfered to the new tube through cell strainer. ->

    Cells are counted in the Neubauer chamber, yield of live cells is normally 1-2*106 cells/mL ->

    Finally cells are placed on laminin and poly-L-lysine pretreated 24x24 cover-slips (concentration is 5*105 cells/mL) and grown at 37C 5% CO2 incubator. ->

    Next day after procedure half of growth medium is replaced with preheated fresh growth media. Same procedure is performed later every 3 days.

    So the question is: We get lots of glia (that we once were suppresing by AraC, but do not do it anymore) but only few if at all live neurons, so does anyone have any suggestions what is wrong with our protocol or what can be improved, or maybe by any chance you do have another protocol that would work for you and that you could share with us? Thank you in advance.

    Yuan Lee · Sun Yat-Sen University

    Thanks a lot ,Enrico Marani.  I get it ~~

  • Yuan Lee added an answer:
    Difference in reagents for dissection/growth of mouse vs rat cortical neurons?

    Hello all, 

    I usually perform dissections and culture postnatal rat cortex (P0-P3) and am about to try out the same dissection in mouse. I use a modified version of the brainbits protocol for cell dissociation (papain, etc). I was wondering if anyone had come across reagents that work well for rat cortical dissections but not mouse? Or additional reagents/issues/anything relevant for mouse, that doesn't come up in rat? Thanks for your help in advance!

    Yuan Lee · Sun Yat-Sen University

    Sorry for the delayed response. Sounds like there is sth, wrong with your culture system. After dissociation, I use DMEM containing 10% FBS as my culture medium. Then 3-5 hrs later, I change the medium with NeuroBasal containging 50* B27 and 400* glutamine. 

  • Kelly Mcgrath added an answer:
    What is the best in-vitro parkinson's disease model?

    Im trying to conduct a neuroprotective assay with SH-SY5Y cells differentiated with RA and TPA to dopaminergic neurons. 

    There seem to be multiple cell lines capable of similar feats, and some that seem to be used more without differentiation.

    Is there any reason to go through the trouble of dual differentiation? or would it be easier and more reliable/reproducible to work with N2A/CATHa/PC12 cell lines, as they seem to be the standard in neuroprotective research. 

    I understand that SH-SY5Y cells are of human origin, but even still, they dont seem to be heavily used when compared to mouse line.

    Kelly Mcgrath · University of Montana

    Fantastic! thanks to everyone that answered, i hope i can successfully apply your advice!

  • Robyn F Cruz added an answer:
    Does S1P1 inhibition have any deleterious (or protective) effects in brain vasculature (of rodents)?

    Sphingosine-1-phosphate1 (S1P1) regulates various molecular and cellular events in different body parts. Its expression pattern and functions are also varied dependent upon the cells types where it is expressed. Does anyone have any information regarding the effect of S1P1 in brain vasculature? Specially in ischemic condition (many paper suggests activation of S1P1 is neuroprotective in ischemic condition, however they lack the discussion on effect of S1P1 in brain vasculature). So if you have any information regarding the S1P1 signaling in brain vasculature, please do share !!!

    Robyn F Cruz · Lesley University

    you have the wrong person -- this is totally out of my area.

  • Victoria Burmester added an answer:
    Have oxytocin receptors been found in the olfactory bulb of humans?

    I am aware that they have been identified in the olfactory nuclei but have they also been found in the bulb?

    Victoria Burmester · Kingston University London

    Thanks Nilesh.  I know it's established that oxytocin receptors are in the rat olfactory bulb.  What I am trying to find out is whether this is so for humans.

  • Tiina Kauppinen added an answer:
    Can I use BV2 microglial cell instead of rat/mice primary microglial brain cells ?

    In most studies the primary microglial cells of the rat brain are use for experiments and I have the BV2 cells ,, So can I use the BV2 cells instead of primary microglial cells of the rat pups brain?

    Tiina Kauppinen · University of Manitoba

    Like previous answers already stated there are differences between BV2 cells and primary cells. BV2s are cell lines and cell lines are programmed to proliferate and thus expression patterns and active signaling pathways differ from primary cells - microglia like any other cells go through maturation/differentiation process (which is quite nicely captured in Prinz & Priller, Nature Reviews Neuroscience 15, 300–312 (2014) ; Fig 2). Phagocytosis relies on various receptor expressions and while BV2 cells phagocyte effectively they might lack the control or selectivity that primary microglial cells posses.

  • Diego Fernández added an answer:
    Why is anisomycin not suppressing my late LTP experiments of synaptic plasticity in the hippocampus?

    I am having trouble to replicate one of the main postulates about the expression of late LTP, concerning its dependence on de novo protein synthesis. I performe fEPSP recordings on the Schaffer collateral to CA1 synapse in rat hippocampal slices. Stimulate with 4xHFS (100 Hz, 1 sec), every five minutes. However, I cannot see an effect using anisomycin 30 µM on late LTP, even if I preincubate the slices in anisomycin for 2 hours before the experiment.

    Villers et al. (PLoS One. 2012;7) already reported no effect of anisomycin or cycloheximide on late LTP. In this paper they argue that the protein-synthesis hypothesis for development of late LTP should be reconsidered.

    What do you think? Do you have any idea about why these experiments are not working?

    Thank you in advance for your help!

    Diego Fernández

    Diego Fernández · University of Vigo

    I prepare anisomycin in DMSO, normally 60 mM aliquates, to be dissolved in ACSF to 30 µM. I tried other protein synthesis inhibitors as cycloheximide, and although is seems to be more effective, the results are still not very reproducible.

    Thank you very much for your helping contributions!

    Best regards,

    Diego Fernández

  • Patro Nisha added an answer:
    Does any one know a good IgG mouse anti nestin to stain neurons in brain sections?

    I am using 40u   free floating mouse brain sections.

    Patro Nisha · Jiwaji University


  • Michele Bastide asked a question:
    Does anyone have a good enzymatic dissociation protocol for mouse cerebral vascular smooth muscle cells for patch-clamp recordings?

    I try to dissociate middle cerebral arteries and basilar artery by enzymatic procedure. Unfortunately, I couldn't obtain healthy cells. They are very fragile and incompatible to patch-clamp   recodings. I used a two-step protocole with first papine and DTT and second collagenase H, collagenase II or F and DTT.

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