Dear Mandy,

I use an Hybridoma against the different fiber type. BA-D5 , SC-71 and BF-F3. They work beautifully. Double check on the hybridoma bank website if they are what you are searching for. I can give you the protocol if you need.

Kind regards

Ornella

As for antibodies I have used NCL-MHCS (dil1:20-1:80), NCL-MHFS (dil 1:10-1:40), M8421 (dil 1;4000), and A4.74 (dil 1;50-1:500).  I have tried multiple dilutions from the suggested ranges on each product. 

This may help,

http://www.sciencedirect.com/science/article/pii/S1095643304001606

Dear Mandy,

I use an Hybridoma against the different fiber type. BA-D5 , SC-71 and BF-F3. They work beautifully. Double check on the hybridoma bank website if they are what you are searching for. I can give you the protocol if you need.

Kind regards

Ornella

Hello Mandy, 

I am not sure whether this helps, but a long time ago, we made a study on fibre types, histochemical staining. See paper  listed below if interested / useful.

KW

Article: Correlation between shortening velocity, force-velocity relation and histochemical fibre-type composition in rat muscles.

K W Ranatunga, P E Thomas

[Show abstract]

Journal of Muscle Research and Cell Motility 07/1990; 11(3):240-50. 

Thank you all for your suggestions and papers! Ornella, would you mind sending your protocol? My email is [email protected].   Thanks again folks. Hopefully I can post a successful progress report! 

Hello Mandy,

I worked for many years in Schiaffino's lab, so I know pretty well the antibodies suggested by Ornella, and I did "some" muscle typing...

I hope that you already managed to get good results with the protocol that Ornella sent you, in any case I can tell you which is the one that I personally developed.

Basically, there are 2 ways, depending on the combination of primary and secondary antibodies that you have.

Let's say you bought BA-D5, SC71 and BF-F3, and your secondary antibody is an anti-mouse, you can use serial sections, and stain each slide with one single primary antibody, and then with your anti-IgG secondary antibody. Then you count how many positive fibres there are per section per antibody, and the total number of fibres. You'll easily end up with a quantification of type 1, 2A, 2B. But you'll struggle with 2X, which are not stained... By the way, you can find an MYH1 (2X myosin) antibody at Developmental Studies Hybridoma Bank, that is called 6H1.

The protocol is very simple (note that BA-D5, SC71 and BF-F3 DO NOT WORK if you perform a fixation with 4% PFA!!!):

1) air dry frozen/freshly cut sections

2) block with M.O.M., 1h at RT

3) incubate with either BA-D5, SC71 or BF-F3 for 45 min at 37°C, in PBS-1%BSA

4) Wash 3x5min with PBS

5) incubate with secondary Ab (note that BA-D5 and SC71 are IgGs, while BF-F3 and also 6H1 are IgMs, so use the secondary antibodies accordingly!), diluted in PBS-1%BSA, for 30-40minutes at 37°C

6) Wash 3x5min with PBS

7) Mount

Usually it is fundamental to identify both positively and negatively stained fibres, so I normally co-stain with laminin or WGA (wheat germ agglutinin).

Of course, if you want you can try to stain with one primary antibody one slide, perform the whole protocol above exposed, block with M.o.M., and then incubate the same sections with another primary antibody, using a secondary antibody with a different fluorescent conjugate... but you have a decent risk of cross reaction!

The following, is my favourite (which is published in the linked Molecular Metabolism paper!!!

First of all, take a look at BA-D5, SC71, BF-F3 characteristics. You'll see that BA-D5 is an IgG-2b, SC71 IgG1 and that BF-F3 is an IgM. So, if you have the chance of buying 3 different anti-mouse, sub-class specific, you can perform a staining in the same slide with all of the 3 primary antibodies together, in one single step. For example, you can buy an anti-mouse IgG2b made in goat, Alexa405-conjugated (blue), an anti-mouse IgG1 made in goat, Alexa488-conjugated (green), and anti-mouse IgM made in goat, Alexa 594. Then if you need to stain the membranes, to identify all the fibres, you can stain for laminin (let's say an anti-laminin generated in rabbit), and for that you will use a secondary antibody anti-rabbit made in goat, Alexa647-conjugated. All the secondary antibodies must have a minimal cross-reaction toward any other (I personally use Jackson Immunoresearch secondary antibodies).

The protocol is again extremely easy, once you have the proper primary and secondary antibodies, of course!

1) air dry frozen/freshly cut sections (no fixation is needed)

2) block with M.o.M., 1h at RT

3) incubate with a mixture of BA-D5, SC71, BF-F3, laminin for 45 min at 37°C, in PBS-1%BSA

4) Wash 3x5min with PBS

5) incubate with a mixture of the following secondary antibodies: anti-mouse IgG2b-Alexa405, IgG1-Alexa488, IgM-Alexa594, anti-rabbit IgG-Alexa647, all made in goat, diluted in PBS-1%BSA, for 30-40minutes at 37°C

6) Wash 3x5min with PBS

7) Mount

I checked all the combinations of primary and secondary antibodies to exclude possible cross-reactions, as you can imagine you need to repeat this with your secondary antibodies. With this protocol, you'll be able to stain beta-slow, type 2A and 2B myosins, while 2X will remain unstained. You'll also be able to find mixed fibres, especially slow and 2A, while you'll find a also many weakly positive 2A fibres, which are often 2A-2X mixed fibres (this you can check by using 6H1 in place of BF-F3). Take a look at one example, following this link: http://www.molmetab.com/cms/attachment/2022349566/2042060853/cover.tif.jpg )

I usually get BA-D5, SC71 and 6H1 as supernatants, and BF-F3 as concentrated purified antibody from DSHB, and use them at 1:100, 1:100, undiluted and 1:100 respectively (since different batches have different concentration, you need to test these dilutions).

The anti-laminin antibody I'm using is the L9393 from Sigma-Aldrich. Having the outline of the fibres is very useful if you need to automate the counting procedure (in this regard, take a look at the paper that I link I provided at the end of this post)

Of course, the conjugates that I suggested are the one that I normally use, but might not be the best choice for your set up. Before buying a particular combination of secondary antibodies, check the filter/illumination combination of you microscopes, so that it is able to visualize and separate the fluorophores! I must say that Alexa305 is not so bright...

I hope that this can help you!

http://www.skeletalmusclejournal.com/content/4/1/21

http://www.molmetab.com/article/S2212-8778%2813%2900103-8/fulltext

Here's an example of what you can get, with the triple staining (which can also be found in DSHB website, in the BA-D5, SC71 or BF-F3 dedicated web page)

Thanks dear Stefano, your info is extremely helpful and I am planning to try it soon. 

 I have a little query, is air drying good for muscle sections ? I kept mine at RT for half an hour and they looked shrunken and horrible. Now I keep them hydrated all the time. 

No, that does not depend on the air-drying procedure, actually I got some staining artefacts when not doing this. Let me ask you, which is the method that you use to freeze the muscles? Are you incubating muscles in sucrose before freezing them? And then, do you use freshly cut sections, or do you use frozen sections?

Thanks, 

I dont use sucrose, I keep them in PBS before snapfreezing with isopentane. freshly sectioned tissues look very nice with hardly any freezing artefact. 

 I use freshly cut sections. 

Stefano, I cannot thank you enough for taking the time to respond to my question! That post was immensely helpful! Thank you, thank you, thank you!

Good to hear that my suggestions are useful, Mandy!

 Dear Stefano, 

 I tried your second procedure (antibodies cocktails, short incubation at 37c) but it didnt work. I have the same 3 primary antibodies from DHSB but while my secondaries (IgG, IgG2b, IgM , all anti-mouse) are from invitrogen. 

I used 5% goat serum plus 1% BSA in PBS for blocking instead of M.o.M (unavailable). 

Muscles were in PBS (no sucrose) and then snap frozen with isopentane chilled with liquid nitrogen. 

I tried both with air drying and hydrated sections (air dried sections look nicer as you said) but it didnt work. 

Any suggestion where I might be wrong ? (my DHSB antibodies are 18 months old, should I order and try with new ones?)

 Loads of thanks for all the help :)

 -- Rizwan

Hi Rizwan,

I guess your staining gave a lot of membrane/interstitial fluorescence, isn't it?

I think your problem is either due to non efficient blocking, or as you said to primary antibodies that are not working properly.

If the problem is due to primary antibodies, you can check them singularly. I suggest to use rat muscles, so that you avoid any confounding effects due to secondary antibody, when using mouse muscles. And you can skip the blocking step. Rat EDL is perfect: you have some type 1 and 2A fibers, and of course many type 2B (which are the majority). Usually DSHB supernatants are stable for a long period, I have some which are still working after 4 years! Probably the BF-F3 antibody (in the concentrated form) is more labile than the other two, and it happened that I had to increase its concentration after some months.

If the problem is due to non efficient blocking, you can try to increase goat serum to 10% (which I normally do).

Good luck!

Stefano

Hi Stefano

 Yes the membranes have strong staining but it doesnt seem to penetrate inside of the fibers. 

 Unfortunately we dont have rat muscles here so I have to stick to mouse. 

 I will increase the goat serum as you suggest and primaries too (i used 1:100 primaries yesterday) and see if it works. 

  Many thanks for the valuable input, many thanks :)

 - Rizwan 

It didnt work with 10% goat serum either and higher concentration (1:40) of primaries. My secondaries work well and the immunoglobin pattern exactly matches with your description. 

Now I wonder if antibodies penetrate the cell at all , may be I should try permeabilization and/or fixation ?

Any suggestion ? 

No, I don't think fixation will help. Actually, PFA fixation is not compatible with these stainings, while cold methanol and or methanol/acetone are suitable. And I never used permeabilization with there antibodies: since you are working on transverse sections the antibodies doesn't have to cross an intact membrane to bind to myosin epitopes.

At this point, I guess the problem is due to primary antibodies not working anymore.

I'm going to order the primary antibodies today and see if that is the issue. 

As ever loads of thanks for your suggestions and taking time out :) 

Stefano, loads and loads of thanks. It worked with the new batch of DSHB antibodies.

 I have to improve on staining and imaging quality but that is easily doable.

 One last question, How do you get such beautiful sections ? For me, I put them in PBS (no sucrose) then freeze with chilled isopantane (the muscle is straight and slightly stretched when I dip it), and then sectioning with OCT embedded around. After sectioning I put them into -80c straight, on the experiment day I did one hour drying at RT before starting the staining. 

 Can you kindly suggest any advice for good looking sections ?

 and big thanks :)   

I already posted some suggestions here: https://www.researchgate.net/post/How_to_properly_dissect_soleus_muscle_for_cryosection

take a look at Partridge's answer, too.

I do not wash muscles in PBS, by the way.

You can embed the muscles in OCT before freezing them. It helps a lot with cutting, especially tiny mouse soleus and EDL. And beware of putting too much OCT, when mounting muscles within the cryostat. OCT is at RT, so when you mount muscles, it tends to thaw them, and then they will freeze again, but quite slowly, since you are around -20°C. And that will create holes in the muscle, even if you initial freezing was perfect.

https://www.researchgate.net/post/How_to_properly_dissect_soleus_muscle_for_cryosection

Many thanks Stefano for very valuable suggestions. I realize OCT in cryostat can cause artifacts. I am planning to freeze with thin layer of OCT and then cut the same without adding more OCT in cryostat. 

 Many thanks again 

Hi Stefano, I am having issues on fiber typing in muscle cryosections using DSHB antibodies (SC-71, BA-D5 and BF-F3). I used the procedure you mentioned except I did it at RT with 2 hours for primaries. Does it matter I did at RT rather than at 37C?

The morphology looked good on H and E staining but was horrible after immunostaining. Also, the staining worked for SC-71 and BF-F3 but not for BA-D5. I used 1:100 dilution for all the antibodies and they were recently bought from DSHB. Any suggestions?

 Also, sometimes I only get staining in the periphery of the sections. Any inputs on why that is happening?

Hi Priya,

2h at RT are fine as well, I don't think this is the problem. I don't even think this can be the cause of the morphological degradation that you observed. In the past, it happened that a colleague received a batch of BA-D5 which was not working properly. To check if this is the case, try to use BA-D5 only, and then a secondary antibody anti-mouse that you know is working for sure. And use mouse or rat soleus muscle, which have many type 1 fibers.

By the way, which is the muscle that you used? If it is a tibialis anterior of a C57 BL/6 mouse, it is quite likely that there aren't type 1 fibers.

Finally, the stain on the periphery of the fibers, is due to unspecific binding of the secondary antibody, if you're working on mouse muscles.

Stefano, If I've already fixed all of my samples in 4% PFA, do you recommend I use a different assay for determining fiber type or are there myhc antibodies that will work with PFA?

Hi Lemuel,

I'm afraid, I have no experience with other commercial antibodies. Just giving a quick look, it seems that Abcam has an antibody for type 1 myosin which works with PFA fixed muscles (ab11083). Obviously, I can not guarantee that it works.

 Hello all, opening this topic back up! Thanks again to Stefano for taking the time to aid us.

My question pertains to the fiber staining in human skeletal muscle, using DHSB antibodies.

I notice many of your stains come out very specific for one antibody. That is not the case for me. Typically, there is a staining combination in each particular cell. One stain in particular usually is dominant, but it is not nearly as crisp as I see in most publications out of your lab. For example, even when stains were not perfect 100% of the time (such as in the recent excellent paper of Murgia), it is still clear there was a distinct stain in each cell for the most part. Mine is not the case. The attached example is a somewhat typical stain for me, however others may not be as adequate. Additionally, it is typical for me to see slight background in nearly ALL fibers (particular for Type 2A and 2X) on individual wavelength channels. What can be the cause of this?

I can provide more images if needed. Thank you again.

Hi Kevin,

separating the channels of the image that you posted, I can see that the blue and red channel are really good staining! You should be really happy with these two!

The fact that the intensity of the staining between antibodies is not identical, is rather normal, and you can adjust the concentration of the primary antibody to make them more similar, or adjust the luminosity/contrast of the entire channel to get a better visualization.

What's problematic is the green one, the ratio between signal and background is not optimal, and there's some interstitial staining, too. Which is the combination of primary-secondary antibodies that you used? And what about muscle and animal?

I can see that there are many speckles, that can be as big as 1/4 of a fiber, especially in the mid-right part of your picture. These look like freezing artifacts to me, or formation of large ice crystals on the top of the sections (maybe formed during the storage in the freezer?).

There's also a cutting artifact, due to the blade, which can be seen with an approximately 160° angle, particularly evident in the green channel, again.

FInally, don't forget that there are mixed fibers, which can be stained with two antibodies!

Let me know, so I can give you a better answer.

Stefano

Stefano,

Thank you for your reply. Yes that is a common problem for us with the green image (6H1, Type 2X). This is one of the nicer stains we are able to get.

Specifically for green channel, we used 6H1 primary antibody with Alexa Fluor 488 goat anti-mouse IgM.

This is a Vastus Lateralis in human.

One hypothesis we have is that, given our subjects were highly trained, the MYH1 might not be readily present in the tissue, or that it may be in low abundances in most of the tissues due to training.

About the cutting artifact, how do you visualize this? A program?

Thank you for your help, I truly appreciate it.

Kevin

Hi Kevin,

I simply used Fiji (ImageJ) to get the 3 RGB channels (Image->Colors->Split Channels), the resulting gray scale images are attached. From the green one you can see clearly see the direction of the blade.

I may say a couple of things about 6H1 antibody. To my knowledge, it is the only one that recognizes type 2X myosin, so there's no other alternative. But, someone from DSHB once told me that they figured out it is a labile IgM, that tends to degrade pretty easily (and that's the reason why you can not buy it as a purified, concentrated antibody from DSHB). So I'm usually using the supernatant at 1:1 dilution overnight at 4°C (or even overday+overnight), in combination with the SC71 and BA-D5. And the staining is usually much weaker than the one obtained with SC71 or BA-D5. Maybe a fresh batch of 6H1 could help to get a better staining.

One more thing about specificity: while SC71 in murine samples is extremely specific for type 2A myosin heavy chain, in human muscles it also stains type 2X fibers. So usually fibers strongly stained with 6H1 (truly 2X), are also weakly stained with SC71 (much weaker than bright SC71 positive fibers, anyway!).

Stefano

Stefano,

That is truly interesting! We've had many hypotheses surrounding the 6H1 antibodies. Is it possible there are actually MYH2 isoforms in a 2X cell? Possibly indicating phenotype shift?

Your answer was very informative, thank you. This comment is more out of curiosity.

Thanks,

Kevin

Yes Kevin,

there are fibers co-expressing MYH2 and MYH1. Moreover, a shift is very likely to happen, especially if training is involved, like you mentioned in your previous comment.

Stefano

This paper might be of some help to you:

Preprint Damaged muscle fibers might masquerade as hybrid fibers – A ...

Good luck!