Dear Liyana,

I am sorry about the problem that you have with the preparation of the material, but there is no other solution than to be patient and work delicately on the surface of the bone. Other way you will destroy important information from the bone surface. You can try to soften the muscles and other remains by placing the bone in a trypsin-buffered solution for a few hours. You have to check the remains on the surface of the bone every other hour, to verify when it is easier to remove them. You cannot leave the bone in the trypsin solution because it can be digested by the enzyme. You have to wash the bone once removed from the enzyme. Thus, as you see, the enzyme will help to soften the soft tissue but still you have to do the work to remove it. I suggest that you do this procedure under a stereomicroscope to avoid destruction of the bony surface.  A time-consuming process in all cases, but it is important to do it properly to ensure good results for histological and/or SEM studies. 

Best wishes,

 Putting it in trypsin solution is an excellent tip. Thank you, Gloria!

Also you can try KOH solution about 10%

KOH is OK, but you also can try it with Trypsin or, more rapidly and radically, with chlor fluid (take care proceeds rapidly --depending on the degree of dilution-- and also results in a certain degree of descaling

If You have the whole information about bone, You have to remove not completely the surrounding tissues in order to prevent the damage of periostium with vessels and mesenchimal cells inside it, and attachment of tendons. For fixation procedure, You have to lenghten the fixation time of about 30-50%. If You are studying only the internal structure of the bone (osteons, trabeculae and other, a patient cutting and schratching of the bone surface is quick and easier.

I do not understand why you would wish to remove all the soft tissue for the examinations you are undertaking. The outer edge of the bone is a complicated soft tissue structure which could easily be damaged by scraping destroying relationships between the bone and periosteum, ligament, and tendon insertions etc. I have always tried to preserve these structures. Histology makes it relatively easy to tell the differences between all these tissue and surrounding muscle etc. I have done no microCT but having seen it carried out on skulls teeth etc by colleagues do you need to rove the soft tissue for this?

If you just want bone - boil it or beetle it but for histo I debulk the muscle from the bone without touching the bone surface and then fix it in formol saline prior to slow decalcification, sectioning and staining.

Dear Colleagues,

There are several ways to do this, but to our experience, using a technique as suggested in e.g. http://discountdermestidbeetles.com/ gives fine results. Afterwards you can further bleach the bones with a peroxide solution (using a soft paintbrush).

Wishing you success, with kind regards,

Koos Jaap van Zwieten

Dear Liyana,

I think that we have not asked you how many bones do you have for this purpose because if you have only one, it should be awful that you are trying different suggestions on it!  However, if you have several specimens (bones) that you can spare, you can try different techniques on different ones and test what it works best for your specimen. But finally, in all the cases, the responsibility is on your hands, because you have to be very careful and overall be patient. Dissections well done are time demanding and require dedication from the researcher.

Best wishes,

Gloria Arratia

You have received good suggestions already.  I suspect just soaking in water (  for x time)   and then very gently picking off tissue with forceps would work.  No quick fix!

bone preparation by soaking in water etc will not allow histology afterwards neither will the beetles.

If you want to carry out histology prompt immersion of the bone with a small amount of adherent tissue in a reasonable volume of formol saline 5 to 10mls for a rat femur will give the best. I am assuming that your intention is for the bone to be decalcified, blocked and cut for staining with H&E etc 

I might need to measure the length and weight of the bones therefore I think I still need them to be completely free of soft tissues. The problem now is how to do that without damaging the periosteum. However I agree with Nigel Harcourt-Brown that for histology complete removal of soft tissues is not necessary. The same probably goes for micro CT as well.

Dear Liyana,

You are expecting a lot if you want to have the bones completely free of soft tissue. Certainly, this point is depending on your dissection's skills. I do not see the problem in taking the length of a bone with some remains of soft tissue, but these remains may affect the weight of the bone itself. What you need to clarify what is the goal of your project and depending of that a few remains of soft tissue (maybe) are not a problem at all or not. At this point, you have received several possible methods helping to clean the surface of a bone, have you tried any of those suggestions yet?

Best wishes,

I have not had the chance to try any of the methods suggested yet but will try them when I do. I really appreciate the suggestions given. Thank you everyone.

Liyana:  I am coming into this a bit late, but the best technique is to subject the bones to 1% solution of enzyme detergent, Tergazyme, at 50 degrees Celsius.  This detergent is commonly used in cleaning surgical instruments.  Tergazyme is made by a company, Alconox, and while they will not provide proprietary information about the enzyme composition, tests over decades have demonstrated its efficacy on fresh bone.  It is very gentle, leaving unmineralized osteoid intact, but removing ALL other soft tissue remnants.  The enzyme must be changed out with fresh solution every day or every other day until clean.  The many rat bones we have cleaned take several days or a week or so to be cleaned. 

Hit Tim,

That sounds interesting. Does it still allow you to carry out histology?