Skeletal Muscle Fibers - Science topic

Hi, I have been experiencing the same with you. Do you find the reason why C2C12 die after differentiation?

Dear Leander Vonk, pardon me:

GOOGLE-searching for ONLY "modified Gomori Trichrome (LG)" reveals a lot of commercial dye vendors (kits, dye powder etc.)... IMHO you need to - at least - specify WHICH particular >Staining Method< out of the presumably at least 10 different "modified Gomori Protocols" you are using to intermittently get different, hence unexpected staining patterns. You should also tell the interested researcher / colleague, which further/additional histochemical reactions in parallel you usually - on a basic routine - will perform - just to evaluate your findings (e.g. MGT + SDH-COX-stain) ... Also, you might have a closer look into other possible stains / staining techniques (comparing stained section images - e.g.: https://www.google.com/search?client=firefox-b-d&q=modified+Gomori+Trichrome+LG#vhid=6u9LL6s1HG8XmM&vssid=l),. If you decide to reply to my post/comment, you might also - please - add an information which sections you are staining: 'wax' embedded or cryosections ?? and also if there might have been variations in the primary fixation (if chemical fixation was applied initially).

Thank you, with my best regards, W. H. M.

Hi Fan,

Assuming that you collect samples at birth or early neonatal stage. There is evidence that animal muscle fiber numbers and myogenesis rates are positively associated with fetal circulating insulin and IGF-1 concentrations near term, and negatively associated with circulating norepinephrine and cortisol. Those correlations also apply to hindlimb mass.

See two papers

J Endocrinol. doi:10.1530/JOE-19-0273.

J Physiol. doi: 10.1113/JP275230

I think that It is better to use a differentiation medium.

Hi Bilal Ahmad Mir it is lifting in the differentiation media, I have used 4:1 DMEM:M199, 2% Horse serum, 1% anti-biotic, HEPEPS, zinc sulphate and vitamin B12 as previously been done. I have not tried other coating, so I am attempting to see if matrigel works. For myoblast stage, they grow perfectly and dont lift.

Using the EVOS FL Color, there isn't a good way to get four channels out of 3 light cubes. You are limited to 3 colors with those light cube options. I would instead recommend the following four dyes/light cubes: DAPI, GFP, Cy3 (or Alexa Fluor 555), Cy5 (or Alexa Fluor 647). But this would require you to have Cy3 and Cy5 light cubes instead of a Texas Red light cube.

Hi.

Where does C2C12 derive from?

If C2C12 once confluent when cultured in myoblast state with 10% FBS, it will never again It will not differentiate to myotubes. I faced the same problem as you, I would recommend making a frozen stock at about 40-60% confluent with ATCC derivatives.Note that some of the commercial C2C12 seems to be mixed once confluent. In that case, give up and try another origin. At least the ATCC ones are sure; the same can also be applied to 3T3L1.

I have tried numerous lots of horse serum and have not found any particular differences between manufacturers and lot. I have never done differentiation enhancement with the addition of Insulin-Transferrin-Selenium because I intended the experiment to include insulin signaling.

Good luck!

I Have encounter the same problem like you. Have you solved this problem?

I also want to calculate muscle fibers CSA in H&E staining using imageJ. Thresholding cannot be used but manually it can be done and it will be time taking process. I want to know about how many and how the fibers are randomly selected in a sample field, how many sample fields will be needed and what should be the magnification?

I am definitely not an expert on this but if you allow the C2C12 cells to differentiate (i.e., switch to differentiation media) and mature, they will spontaneously contract. I have done calcium imaging on such cells and they light up like fireflies as they contract.

Isabella Bagdasarian hi thanks so much for this! I tried this recently but now have a few follow-up questions. What do you dilute your APTES in? I've tried another compound, DETA (N1-(3-Trimethoxysilylpropyl)diethylenetriamine), and used 1% in toluene at room temperature for 2 hours. Do you heat your solution? And for how long? Is this necessary, and do you use a hot plate for heating it?

I then did a wash in toluene before curing in the oven, but there was a lot of residue on my coverslips from the treatment. Unsure if I did something wrong, I mostly followed protocols I found in papers. Don't think we can do contact angle goniometry to see if it worked so mostly looking at how the cells respond and my 12-well plate was a mixed bag of results, but none of them lasted 7 days, never mind 2 weeks! Do you have a detailed protocol written for someone who's never done this sort of treatment before? Really appreciate the help, thanks so much again!

This is out of my area of expertise but I have read in numerous papers where application of cyclical uniaxial stretch to muscle cell cultures results in the myotubes being parallel to each other. From what I understand, there are commercially-available devices for doing this.

We tend to initiate differentiation at a slightly lower confluence (about 90-95%%) and we too, switch out the differentiation medium every 48hrs with warm medium. Myotubes, as you said, are usually fully formed in 4-6 days. When the confluence gets too high, you can get early differentiation as the myoblasts start to fuse together and the media conditions aren't optimal at that point, leading to smaller, less differentiated fibers (so we don't usually go all the way to 100%)... and there is just less room for the fibers to grow and extend out.

I didn't see it in your protocol, but we also found that many of our cells do not adhere well to normal plastic cell culture plates. We coat our plates/wells with gelatin (0.1% gelatin (sigma G9391): 0.5 g gelatin in 500 ml ultra-pure H20, autoclaved at 121C, 15psi for 45 min, store at 4C) To coat our plates, we warm the gelatin solution to 37C, and add just enough to each well/plate to cover the bottom of the plate. We incubate at room temperature (in biosafety cabinet) for 1 hour and aspirate excess gelatin. After the plates completely dry, they can be stored for up to one month at 4C (though they should be warmed to room temperature prior to adding the myoblasts that will be differentiated).

I hope this helps with your differentiation endeavors, good luck!

After differentiation we continue to change the media every other day (48 hrs)... also, we never go 3 days between changes... if the 48-hr media change falls on a weekend, then we come in for a quick media change on the weekend...

These journal articles should address what you have requested:

Laumonier T, Jacques Menetrey J. Muscle injuries and strategies for improving their repair. J Exp Orthop. 2016;3:15. doi: 10.1186/s40634-016-0051-7.

Karalaki M, et al. Muscle regeneration: Cellular and molecular events. In Vivo. 2009 (Sept);23(5):779-796.

L. Baoge, E. Van Den Steen, S. Rimbaut, et al. Treatment of skeletal muscle injury: A review, Int Scholarly Res Notices. 2012; doi: 10.5402/2012/689012.

As the previous answer suggests, there seems to be contamination, I have routinely differentiated C2C12s in the presence of penicillin and streptomycin - you should try this out (50 i.u. penicillin and 50 μg/ml streptomycin). Equally, it is important to evaluate the cells daily and change the differentiation media every other day.

Muscle cell cultures are already in an atrophy state, i.e., there is minimal (if any) contractile loading. Perhaps it would be better to go in the other direction, i.e., increase contractile loading, so as to promote "hypertrophy". Electrically stimulate the cell cultures. This is harder than it sounds. Too much stimulation can kill cells.

Erica Hurtado I, myself, would go with the tibialis anterior. For one, we use it all the time and we never use the gastrocnemius. Second, the attached article by Burkholder et al argues for it. Fibers are longer in the TA (see Table 1) and thus it should be easier to get away from the neuromuscular junctions when you want to induce injury. Also, the TA architecture is more fusiform in nature than is the gastrocnemius (again see Table 1). With a simpler geometry, I think that it helps make it easier to stay away from the muscle fiber regions with most of the neuromuscular junctions.

Nice question but not sure if well size affects the process of differentiation. I believe that it should not as cells are grown to more than 80% confluence and then starved or incubated in reduced serum media. Irrespective of size, cells will grow to a certain confluence and then differentiate upon reduction of serum. I have tried different size dishes, flasks and 6, 12 and 24-well plates for both primaries and C2C12 cells, they have differentiated similarly. I

I agree with Patrick Davis but I have never tried smaller well-plates.

Sahar Salehi-Müller

Thank you for your comment.

I cultured C2C12 cells in 6 well plate with 3 ml medium when I differntiated them (Before the differentiation, I used 75cm² flask with 15 ml medium).

I think it could be the lack of nutrient or not because I seldom saw the small cells in the 75cm² flask.

myotubes had significantly larger diameters and more total sarcomeric myosin expression than Park7 (-/-) myotubes

Hi Angelica Benaim,

You can split or merge the channels using ImageJ (Fiji). You can find some instructions here:

https://imagej.net/Color_Image_Processing#Merging_multi-channel_images

Regards

Hi Chukwuweike Uchenna Gwam

Hi Andrew,

It's many years since you posted this question, but I'm having similar difficulties at present and wonder if/how you resolved this?

Best,

David

Thanks Frederic for the valuable input.@Frederic N Daussin

Thank you very much. I will see it at once.

I really appreciate your reply.

kazuma @nagahata

Dear Jj,

you can improve the C2C12 attachment by pre-coating your dish/wells with a suspension of Matrigel diluted 1:80 in DMEM.

Regards

Iwan Jones Thank you for all your help, I will try some of these things out!

Dear Alessio,

Thank you very much for the very important information.

Best

We have been measuring human muscle forces during orthopaedic surgery, directly at their tendon and as a function of joint angle after stimulating them. Hence, we obtain muscle force-joint able characteristics, from which muscle stiffness can be determined in active state. We also measure relevant anthropometrics before surgery, including circumference of the limb and tendon cross-sectional area if relevant. One finding which is very striking is that human muscle forces vary a lot from participant to participant despite their similar conditions. One aim we have is to see if such pre-surgery anthropometrics are good metrics to characterise the target muscle. However, there is almost never a correlation between e.g., the peak force and those metrics. In one particular study (which is attached), we looked to see if tendon stress instead of tendon force can remove the variability. In order to achieve that, we calculated tendon cross-sectional areas of the participants and determined the stress as force/cross-sectional area. This did limit some of the variability but did not eliminate it. We have been measuring forces of spastic hamstring muscles of cerebral palsy patients to characterise the pathological knee condition. However, such anthropometrics and the actual muscle force do not seem to correlate. I will attach one recent example of such intraoperative CP work as well.

Use as differentiation media: DMEM supplemented with 2% horse serum and change the media every 2-3 days. Try to use a support like Matrigel and start differentiation with about 80% of cell confluence.

It is possible to do flow cytometry of myotubes until unless you make sure that there are no clumps. You need to adjust the forward scatter to see the cell populations. And it is better to have low cell density in the samples. See the following paper for more details.

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Hello

Kindly look at links here 

Hope help you

Good Luck 

I agree with Denise. In my case It has been about twenty years since I isolated satellite cells to grow but as I remember it involved digestion and d damage to the fiber (myocytes). Can you use fluorescent or immune fluorescence microscopic approach to both distinguishing the satellite cells from the myocytes and also for measuring drug uptake?

You may consider using myogenic stem cells and creating in culture myofibers (myotubes). I have been inducing myogenesis in C2C12 mouse myoblasts quite some time, it is easy.

Hello, do you have the protocal of ATP staining of skeletal muscle, I want to have a try of this experiment, thank you!

Contact the author directly in Paris.

The Triton X-100 is used solution in 1% vol/vol, in relaxing solution with EGTA 5mM, for 10 to 20 minutes. In this case the results are very goods.

Dear Gayoan,

I think it's very simple—the VL is big and easy to get a biopsy from with very little risk of injury.  This is the key muscle for human research primarily for that reason.  The human fiber type argument is weak.  Look at our paper regarding human fiber types and you will see that all of these muscles are pretty slow:

Human skeletal muscle biochemical diversity.

Tirrell TF, Cook MS, Carr JA, Lin E, Ward SR, Lieber RL.

J Exp Biol. 2012 Aug 1;215(Pt 15):2551-9. doi: 10.1242/jeb.069385. Erratum in: J Exp Biol. 2012 Aug 1;215(Pt 15):2931.  PMID: 22786631

I hope that helps.

Rick

Dear Shafagat,

Thank you so much for your information. Actually, I already know these links, but I don't know where could I buy the T-clips.It is said only a small company has that product, while I am not sure which company it is.

Sincerely,

Gaoyan

I think the strict answer to your question is that no one really knows.  Quantifying satellite cell number, especially with exercise or degeneration models is really challenging.  More SCs in Type 1 fibers is pretty much agreed upon, but not universal.  Also, with exercise, many of these changes are transient.  I would take a look at Mike Rudnicki's latest review:  

Satellite Cells and Skeletal Muscle Regeneration.

Dumont NA, Bentzinger CF, Sincennes MC, Rudnicki MA.

Compr Physiol. 2015 Jul 1;5(3):1027-59. doi: 10.1002/cphy.c140068.

PMID: 26140708

Yes, it seems that many groups have studied the "aerobic" models of in vitro myotube stimulation, but I would check out the following paper from Keith Baar's lab: http://www.ncbi.nlm.nih.gov/pubmed/19807268. They were able to modulate the frequency to more of a "resistance" model, and demonstrated an anabolic like response.

David Hood's group at York (Canada) has also completed quite a bit of research in the in vitro e-stim area, although they focused more on mitochondrial biogenesis. You may want to also check them out for some experimental designs.

Hope this helps. Best of luck.

dear Shakil Urrehman

i hope next site help you

best regard hamdy

Honestly, I think this is a lost cause.  First of all, the two collagen groups are intimately intertwined so that there is probably no functional distinction.  Second, there are no collagen types that have been uniquely associated with one or the other.  These are "convenience" terms for us and probably have no true biochemical distinction.  The only thing that we have found that is unique about the perimysium is the the large collagen bundles.  For endomysium, you could probably use basement membrane collagen type IV just to see where it should be, but it will not define the extent of it.  Good luck!

Dear Antonio,

I dont know any specific references, but I would recommend you to check work from Simon Gandevia from UNSW, Sydney Australia. He and his collegues have 100s of electrophysiological studies in humans. If you go through their papers you may find alot of usuful information.

Best wishes,

Refik

My lab has developed Mabs against 2B and 2B MyHCs (Lucas CA, Kang LH, and Hoh JF. Monospecific antibodies against the three mammalian fast limb myosin heavy chains. Biochem Biophys Res Commun 272: 303-308, 2000). They work even in marsupials (Zhong WW, Lucas CA, Kang LH, and Hoh JF. Electrophoretic and immunochemical evidence showing that marsupial limb muscles express the same fast and slow myosin heavy chains as eutherians. Electrophoresis 22: 1016-1020, 2001), and should work in bovine tissues. They are MAbs 6H1 and 10F5 (available from Developmental Studies Hybridoma Bank; University of Iowa, IA), specific to 2X and 2B MyHCs, respectively. They work in Westerns. Good luck with your work, Cruz.

Folch will most likely extract glycolipids, but it would be interesting to be sure by using TLC of the extract: some lipids could potentially remain in the interphase between the 2 solvents after extraction. 

As far as the amount is concerned, it strongly depends on the method you are going to follow subsequently for glycolipids analysis. The amount of gangliosides and cerebrosides in muscle must be quite tiny, as far as I can remember from out TLC plates when analysing pork phospholipids.

The amount of sample can be increased, as long as you keep the sample to solvent ratio:

 https://www.researchgate.net/publication/222271833_Comparison_of_different_methods_for_total_lipid_quantification_in_meat_and_meat_products

Very interesting, thank you!

Hi Robert,

How do you currently separate and permeabilize fibers?  Also, what species are you studying?

Regardless, I would recommend watching this JoVE video for how to separate fibers:  http://www.jove.com/video/2431/respirometric-oxidative-phosphorylation-assessment-saponin

The Oroboros website (http://oroboros.at) is also an excellent resource for both protocols and theories relating to respirometry.

Hi Anna,

If I were you I would go for soleus and EDL to start with.  These muscles are of similar size.  Personally, I would not determine MyHC isoform changes by immunocytochemistry.  A more precise and reliable method in my experience is to separate MyHC isoforms on SDS-PAGE .

Good luck

The above assumption is logical. However, the literature is not enough data to draw conclusions. It is possible to partially solve the problem of muscle growth with a lack of methylation Supplementing the necessary amino acids.

Hello Ponmathi

I used to do this for the majority of woman who I saw, with pelvic floor symptoms; I used the method that Jo Laycock and colleagues designed and validated. I don't know if you've seen this method - I was introduced to it by Jo, and never changed from this.

Laycock, J., & Jerwood, D. (2001). Pelvic floor muscle assessment: the PERFECT scheme. Physiotherapy, 87(12), 631-642.

Part of this is available from ResearchGate:

You might want to read a previous ResearchGate Question/Answer where other members also made suggestions:

The other paper I mentioned in this answer was:

Jeyaseelan, S. M., Haslam, J., Winstanley, J., Roe, B. H., & Oldham, J. A. (2001). Digital vaginal assessment: An inter-tester reliability study. Physiotherapy, 87(5), 243-250.

If you are unable to access the full text, I have a copy  that I could send you on ResearchGate messages, for your attention only.

Very best wishes

Mary 

Hi Emily, 

I hope that by now you solve your problem. Sometimes people have resources close to them and they don't know. This is just in case you have this method available: 

To count by eye might become an extenuating task. However, if you have a 488 nm laser available in your system (together with the transmission set up) you could take an image under "transmission" (without the need of adding any fluorophore or  additional compounds). This way you could save tons of images and analyze them later. 

 

Good luck,

Carlo

There is a good reason why the original Goldspink paper has not been followed up - it is a difficult system.  The suggestion of tritium label is probably the best way of getting high resolution data because of the short range of radiation emission but this is also a drawback because it lowers the sensitivity - in tissue most of the emission decays within 2µm, so much of the signal never gets into the emulsion from standard wax or frozen sections.  This can be improved by cutting thin sections in acrylate embedded material but this requires a long exposure and, as implied by Dr Morgan, noise is a problem that you need to minimize by keeping the exposing slides in lead boxes and by meticulous darkroom work.

The suggestion of using antibody against developmental myosins may work but I would think that it would be technically possible nowadays to produce a mouse with a conditional expression of a tagged actin, myosin, or other sarcomeric protein that could  be activated prior to eliciting regeneration.  The Pax7Cre-ERT system would be suitable for regeneration experiments. Whether you could get funding for such a system is another matter.

At a low resolution, one of my colleagues has tried to use pulsed Stable Isotope Labelling in the Mouse (SILAM) and analysis of the ends versus the middle of the muscle fibres by Mass Spec. So far, his produced very variable results.

You may wish to have a look at the manuscript of Farina et al. 2007 (see attachment).

Hi Umar,

In my experience, hyper contraction of the fibers is always linked to the collagenase digestion. If the digestion is too strong the fibers exiting the muscle are/become hyper contracted. On the contrary, if the muscle is not digested enough you damage the fibers when trying to dissociate the fibers with the Pasteur pipet and the fibers become hyper contracted. To know if the digestion is ok, you should see alive fibers coming out spontaneously from the digested muscle.

I don't know if your muscles are too/not enough digested but you may consider to modify the time of digestion (30->90min) and/or your incubation conditions (agitation in water bath strongly increases the efficiency of the digestion). After the digestion, I also used to transfer the muscle in DMEM 10% FBS and to keep it for 1h in the incubator (an advice I got from Zammit lab). 

I have also heard that high concentration of serum may induce hyper contraction of the fibers. I don't know if it's true but I have always let the fibers in DMEM 10% FBS for few hours before changing for a proliferation medium (DMEM with 20% FBS, 1% chicken embryo extract).

Good luck

Theoretically, the answer is yes. However, I don't know of any evidence that has directly linked the speed of hamstrings tension development to ACL injury incidence. The concentric hamstrings to concentric quadriceps peak torque ratio is believed to be an important indicator of the ability to co-contract the antagonist muscle groups, which is necessary for dynamic knee joint stability. We have demonstrated that this ratio is improved by plyometric training of female college basketball players. Although the attached 2004 report did not include "time to peak torque" data, we have observed faster peak torque development in the hamstrings as strength improves. Some experts emphasize the importance of eccentric hamstrings strength for protection of the ACL, but jump landing requires the quadriceps to eccentrically dissipate ground reaction force (while the knee is flexing). Logically, any hamstrings tension that is simultaneously generated at the knee must be concentric.

Please get the childs full thyroid profile before deciding if hypothyroidism is responsible or not as although thyroid has a role in differentiation in early stages first you have to know if there is hypothyroidism or not and if congenital hypothyroidism is it primary or secondary and further management only comes accordinglyand one tries to see which gene deficiency is responsible and does the child need replacement L-THYROXINE THERAPY OR NOT and get the CRP levels as well to further decidethe management.

This is related to the individual differences (in humans they are significant) and very small sample sizes. Increasing the sample size would decrease the SD and show more valid results.

Yes the difference is molecular weight. I use sodium succinate 6 H2O

it depends on what is the result or function you focused on.

Hi Valeria, thank you for your suggestion and comments.

Hi Mohamed, I can't upload pics, because it is unpublished data. 

Hello,

You may also consider incubating the muscle bundle in "skinning solution" (active ingredient potassium propionate/propionic acid) to poke holes in the sarcolemma (see attached paper for recipe). We do this for isolating single fibers from human tissue samples to investigate contractile properties. After 5-7 days in solution (at -20 degrees C) the fibers pull extremely easily and the microstructure is still intact. 

Hope this helps,

James

If you are using medians, that indicates your data are not normally distributed. In this situation, you should use a Kruskal-Wallis test; it is the nonparametric equivalent of a 1-way ANOVA. A Bonferroni-adjusted Mann-Whitney test is the appropriate post-hoc test if one is needed.

An alternative to using the Kruskal-Wallis test is to see if you can transform the non-normally distributed data to data that is normally distributed. Log transformations are good to try but you might try square roots, et cetera. If after transformation the data are normally distributed, you can use a 1-way ANOVA on the transformed data.

We did measure human skeletal muscle telomere length by TRF assay. As far as I know we used standard published protocols for DNA extraction, digestion and Southern Blotting.

I have figured a semi-automated method using ImageJ. It requires a cell border stain (i.e. Laminin, dystrophin). I will post ImageJ functions shortly.

In my opinion the best "indirect" blood markers of muscle damage are Creatine kinase (CK) and (Mb). Although they peak at different time points (Mb: 6hrs after; CK: 1 day after exercise).

I prefer to quantify the muscle damage using the fluorescent (loss in dystrophin staining) or electron transmission microscopy (Z-disk streaming). Have a look to this article. Macaluso F, Isaacs AW, Myburgh KH. Preferential type II muscle fiber damage from plyometric exercise. J Athl Train. 2012 Jul-Aug;47(4):414-20.

Hi Luisa. If you are intending to carry out analysis of fibre size you might want to consider using Feret's inner diameter (instead of cross-sectional area) which, coincidentally, both Florian Bentzinger who answered your question above and myself have used in our studies of muscle regeneration in the past. More specifically to your question you could then quantify the number of subsarcolemmal nuclei present in your fibres and determine a potential relation myonuclear accretion in relation to fibre size or number (I am not sure what species and muscle groups you are using for your work).

Dear Siacia Broos,

since your question had only one reply very recently since January 2013 I would like to perhaps help you with some suggestion(s). The problem is that there are a lot of papers dealing with Muscle fibres and SEM, but more/less related to either "general" aspects of view or "Localization of Neuromuscular Junction regions", but not specifically doing fiber/fibre typing by Scanning Electron Microscopy. I was not able to localize any article / paper related intimately to your request (also searching PubMed for instance) .

The Reference given by my Fore-Poster Anthony Ciccone seems not to be what you really are looking for.

So the only help I can offer is (this might be turn out to create hours of search and "severe" documentation work, I know (;-)) ) to search Google for the following search phrases (as you can see, number of results decreases with limitations in search phrase):

GOOGLE Search:

< muscle fiber typ* or classification AND Scanning Electron Microsc* > 3.130.000 results (look always for "fibre" and "fiber" too),

< muscle fibre typ* or classification AND Scanning Electron > 2.590.000 results, "skeletal muscle fibre" typing AND Scanning Electron Microsc* 414.000 results,

< muscle fiber typing AND Scanning Electron > 254.000 results,

< muscle fibre typing AND Scanning Electron Microsc* > 158.000 results. I have seen that there are some interesting articles also dealing with fibre typing before/parallel to EM (e.g. TEM, but sometimes SEM as well) but I guess that it will be hard to find any article dealing with fiber/fibre typing/classification only by SEM-technique). If you the find the one or other article which fits into your original request profile, then you might choose other, more specified search phrases (for Google as well as PubMed)...

You have not said anything about the techniques you would like to use for your task: i.e. e.g. only "pure, classical SEM of fibres", freeze drying (FD) or Freeze substitution (FS) and / or freeze fracture (FF)-preps" etc., the latter I think would be necessary to localize a Z-band (you know differences between fiber type-associated Z-Bands already? what about preparatory conditions to prevent artefactual contracture of muscle fibres??, etc.).

What I only could imagine would be: a) FD-FS-FF, b) having many looks into your fibre surfaces and Z-Bands with documentation c) afterwards reembedding your small samples into either paraffin and/or resin, cutting histological sections and/or semithins, IHC-stains for fibre types (with these you could see at least some kind of fibre types/fibre type patterns) & ultrathins for correlative TEM). Another way round would be perhaps (as a trial) Pre-embedding IHC of fibre types, different gold labels and examining FD-FS-FF-specimens by SEM-modes (BSE, SE, etc.).

AS concerning your question <Does anyone know how you can determine fiber type before> please consult major handbooks in (diagnostic) Muscle Pathology. Best regards and good luck!

Xiufang, I'm sorry I can't help directly, but if you're unable to find the antibodies you need, we discover new DNA aptamers as an alternative. Please see www.BasePairBio.com for more information. Best, Bill

Hi Peter, We freeze our human muscle in liquid nitrogen cooled isopentane. Never liquid nitrogen..the freeze damage is too extensive in our experience. We actually cool the isopentane till its just starting to freeze over on the bottom before we drop the muscle in. It's important to get good quality isopentane with low water content...And dont reuse the isopentane too much. Colleagues of ours actually mount the muscle on a small strip of card, as human muscle can be quite soft (depending on what muscle is biopsied and from whom it's from, age, lipid content etc etc) and plunge the sample into the cooled isopentane by holding the card with tweezers. That way the muscle is always orientated correctly and doesn't deform in an undesirable manner on freezing. I havnt done this myself, but it seems to work well for them. I dont have too much trouble just freezing the muscle by itself but It might be somthing to try if you are having issues. Just before freezing, carefully blot moisture from the muscle with some fine grade tissue to prevent ice crystal formation on the surface of the muscle then immediately freeze. We leave the sample in there for 10 to 20 seconds before removing to a cryovial in dry ice. As Clare mentioned, agitation by swirling the isopentane gently seems to help. remove sample with isopentane cooled tweezers. Cryovials are best for long term storage rather than standard laboratory tubes..so specimens don't dry out in the freezer -80oC. Separate tweezers for handling frozen and unfrozen muscle too is always handy! For sectioning, we mount on the chuck with minimal OCT. the chamber and chuck are cooled between -20oC to -25oC. We cut sections between 5 to 8 microns. Use a fine brush to remove any ice crystal formation on the blade (from breath). We mount on super frost plus slides.

Ron Allen (Arizona) has done alot of satellite cell/progenitor cell isolation from rats - you may want to check his papers.