I have not worked with bone but 100um is usually quite deep for confocal imaging, in my experience. Usually i work with mouse skin and i cannot reach such a depth in most cases. Usually is not because the staining is not penetrating enough but because light (specially green fluorescence) is retained in tissue and cannot easily reach the surface. You can always try to permeabilize a bit with triton (0,1% in PBS or PBS BSA) for 20 min. In my hands this can also help sometimes. Also i suggest you to use antibodies that can be observed in IR channels. Anyway i have never work with such a tissue and i don't really now if it would work. Hope it may somehow help you.

Thank you, Alvaro. I’m using upright confocal with 25x0.95 dipping water objective which can penetrate deep into tissue. My z stacks are deep enough, just the blood vessels don’t look perfect. In the first couple of experiments, I didn’t block before staining and ended up with lots of noise as deep as bone marrow.

It’s a good idea to use another chromophore in the red region – I never thought about penetration of laser sources. Actually I used several antibodies and chromophores including CD31-Alexa 647, and need to compare them properly for differences.

Permeabilization using Triton x-100 is also part of my staining.

Thank you, Mary. Is there any advantage of staining for elastin compare to CD31, lectin or SMA in bone? Do you expect more elastin than the other proteins in blood vessels?

About vacuum penetration, I check how feasible it is for my case.

I'm looking for a special type of cells close to the blood vessels and trying to keep the protocol as simple as possible, the protocol I'm using worked in other hands and there are publications for it.

My goal is to get some images as support for our FACS data. Can I still image periosteum after demineralization and collagenase digestion?

Thanks, Mary. I'll try deminerilization to see the difference.

I've never done it in mouse, but I know several people who have used DiI as a Lipophillic Carbocyanine Dye that has really nice fluorescence and will mark even the smallest of capillary vessels. I use it for vascular visualization in rat brain, but the article below uses it for retinal vasculature. It's a perfusion dye procedure and is VERY easy to do. Also if you're looking to go further into the IR spectrum for better optical penetration, you can go with DiD or DiR or for shorter wavelength excitation/emission DiO.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2811090/

Thank you for introducing this interesting paper, Michael. Images are so clear. Just a technical question:

I need to stain bone with primary and secondary antibodies, which take couple of days. Do you think DiI stays in endothelial cell membranes? Isn't there any diffusion problem? This is the case for suspension cells labelled with DiI.

Sorry for the delay in replying, I've been recovering from surgery the last few days. I would be concerned with the IHC aspects interfering as the DiI is alcohol soluble. I presume you may be using alcohol solutions in your processing for IHC is that the case? Also the DiI DOES have some aqueous solubility issues. I use it in over short time scales to evaluate vasculature within 36 hours of perfusion. Over time it can diffuse through the tissue and lose resolution. You may be able to do two imaging sessions though. One with DiI for vasculature and another for the specific antibodies(not sure if that would be applicable in your work?). Best of luck with it!

HOPE YOU GET WELL SOON, Michael and thank you for your points. I'm not using alcoholic solution, but I need to do imaging in one go and cannot split it in two sessions.

This method looks promising, maybe not perfect in my case, AND I'll give a try. I'm sure it comes useful in other cases.

Hi Maryam, this thread is already quiet cold but maybe this information is still of interest to you: The company I am working for has developed an anti-mouse CD31 antibody that to our knowledge is the only one that works really well in standard FFPE sections. For more information and some pictures check out this link:

http://www.dianova.com/en/products/product-highlights/murine-cd31-clone-sz31-formalin-fixed-paraffin-embedded-tissues/

I missed your answer, sorry!

Actually I've been using anti-mouse CD31 antibody for staining new blood vessels in matrigel recently and got very good results. My plan is to stain some bones by Easter.

The difference this time, I have 4 um slices of bone and Zeiss Axio Observer Inverted Widefield Microscope which enables me to get good tile images, as well as confocal.

Decalcification is the next step, if this doesn't work.

Chek this: http://www.jneurosci.org/content/27/22/5976.long

Is for the brain, they use gelatin and BSA-rhodamine perfusion

thank you. staining looks great. Definitely consider it in any future experiment.

Hi..I am going to do immunohistochemistry of cornea to see neoangiogenesis and leukocytes infiltration in corneal suture model of mice. Could anybody suggest me what would be the best stain for this ?

Hi Dinesh.

CD45 is pam-leukocyte marker. 

For neoangiogenesis you can use  von Willebrand Factor antibody.

I also used CD31 (endothelial marker) and lectin.