Superficial fibers in vivo not treated with anything.

thank for your replay.  

thank you very much I think I will give a try the isolated single fibre.

I am assuming you are using fixed tissue.  Try not to use collagenase.  Maybe dilute sulfuric acid.  We and others have done this many times in animal models.  Please see:

 Burkholder, T.J., B. Fingado, S. Baron and R.L. Lieber.  (1994).  Relationship between muscle fiber types and sizes and muscle architectural properties in the mouse hindlimb.  J. Morphol.  221:177-190. PMID7932768

Don't forget to normalize by sarcomere length!

Hi Richard

yes, I had not any success with the collagenase as I have dissected and snap freeze the muscle samples 2 months ago.

I will give the sulphuric acid a try.

thanks a lot.

Good luck!!

For some reasons the sulfuric acid did not work as excepted. I will increase the concertation to 30% and time of incubation to 4 hours.   

 30 % worked nicely I am wondering about how to pull the fibers and how to make sure you are at the right end this is another challenge now. 

Hello,

Many many years ago, we used to do this using rat muscles. Since they were coupled with mechanical force / length recording as well to determine Sarc L - tension relation, The procedure adopted for fibre isolation had several steps, as I recall, given in p299 in paper below.  May be you can check in the paper below (1984, muscle & nerve), if interested..

Regards,

KW

.........................................................................

TEMPERATURE SENSITIVITY OF TENSION   DEVELOPMENT IN A FAST-TWITCH MUSCLE OF THE RAT

 MUSCLE 81 NERVE 7:298-303 (1984) 

MURTADA H. ELMUBARAK, MBBS,

and K. W. RANATUNGA. PhD

(Abstract). Isometric contractions of the biceps brachii (short head) muscle in the

rat were recorded in vitro with direct stimulation and at different temperatures

(range, 35°C-10°C). In confirmation of our previous findings from fast extensor digitorum longus and slow soleus muscles, the time and rate parameters of the twitch and the tetanus showed an increased temperature sensitivity below 20°C. The dependence on the initial muscle length of the rate of rise of tetanic tension was examined at 27°C and at 15°C. When represented as a percentage of the tetanic tension at each length, the rate of rise was independent of muscle length at both

temperatures. Our interpretation of this particular observation is that the increased cooling depression of the rate of tension rise below 20°C is not associated with a qualitative change in its underlying basis.

Dear Ranatunga

Can you kindly send me the paper?

Many thanks

Hi Abdullah,

It should be in my Reseachgate files and is attached now. Hope it is the right one and you can get it!

Regards,

KW R

Dear Ranatunga

Thank you for sending this I am wondering about how long did the muscles incubated in nitric acid.

"  The muscles were macerated in 25% nitric acid and stored in 50% glycerol. Between 6 and 10 muscle fibers, or bundles of muscle fibers containing up to about 6

fibers "

thanks

I just checked Murtada's thesis and a paragraph is copied / attached below (hope readable). Since fibres / bundles are already fixed at opt L, maceration is to separate them. So, no exact time but long enough and some preliminary tests would be useful.

KW R

42 years ago we have suggested the method of alkaline dissociation of formalin fixed tissue (article in Russian): Article [Preparation of isolated cells by alkaline dissociation of f...

I guess that you can use this simple method. I'll be glad to answer to your questions.

When you are first developing methods, you can also use an elastin or collagen stain to make sure you get all the way to the end of the fiber. Honestly, it really depends on the level of resolution you need in your fiber length measurements. In cases where we needed very high resolution fiber length measurements, we include sarcomere length measurements. What do you need to measure fiber length for>