Are you preparing specimens for TEM or optical microscopy?

Are you embedding the whole bone (not advisable for Spurr)?

Dear Milena,

quality problems may occur as you have the soft bone marrow close to the very hard bone tissue. This could be due to issues of infiltration, polymerization or sectioning. You work with 6 month old mouse bone, which is very robust and tough to section.

1. The embedding procedure is routine in the lab? If not, contact someone with more expertise - hard tissue histology is challenging.

2. Check bone marrow for cavities (areas without or soft plastic). This may occur by unefficient infiltration or polymerization (shrinkage artefacts). If there are regions without or soft plastic test in a bone specimen the infiltration and polymerization procedure with new reagents.

3. Consider the slicing procedure. From your description I assume you section long bone. Take a new sharp knife, check the sectioning angle, check the sample holder. Perform sectioning with someone experienced together. Change the direction of sectioning e.g. from distal to proximal or proximal to distal.

If you wish send me an image of your slices and a photograph of your block. Then I can comment a bit better on that issue.

all the best,

jirko

Hi Milena,

Jirko covers pretty much everything. A few things though:

Are you using a tungsten steel knife on a heavy duty microtome?

The thickness we section at is normally 5um, and this works OK for us.

We normally use a methylmethacrylate embedding which polymerises at 30C.There are also protocols fro polymerisation at much lower temperatures. For us this works fine and keeps the TRAcP staining alive. Higher temperatures may affect the TRAcP enzyme.

On onee thing I agree very strongly with Jirko: if your lab does not do this on a routine basis, visit a lab that does. This can save you years of trying to reinvent the wheel.

best wishes,

Rob

Hi Milena,

                  I used to do a lot of resin work and this kind of problem was usually caused by the final consistency of the polymerised resin. You seem to have a good protocol, so assuming that you are not having sectioning issues, the only possibility that I can think of is that your resin is too hard or inflexible. I know a retired gentleman in the UK who is an absolute genius with resin histology. His name is Neil Hand, formerly of Nottingham Hospital, but he is still an assessor for UKNEQAS ICC and may be contacted through them I'm sure.

Best of luck,

                       Kieran

I have used decalcified mouse bone and embedded in paraffin.  If you don't have to cut very thin sections, this might work. If the bone is decalcified for a few days in 10% EDTA in 0.1M Tris HCl pH 7.4, it will be soft enough to be wax embedded and cut on a microtome.

Paraffin bone tissue has to be decalcified. It can not do dynamic histomorphometry. Try   

Jirko and others suggestions. They cover pretty much all you need to do. We use MMA mixture and only dehydrate for total three hours from 90% to 100% acetone before infiltration step mouse long bone

Sorry to be chiming in so late.  Answers above all on target, which require of you some vigilance with the method and more experience.  Best, however, to stop using that resin.  You will all but eliminate the problems with PMMA, assuming you have ordinary diamond saws.

I am working now with MMA, and it works great. Sections look very nice. Thank you all for your answers and help.

Does anyone know silver technique to visualize nerves in Undecalcified  rat Bones?