Dear Allesio,

I work on retina and fish tectum, bot of which are fairly thick. I use imaging chambers. The trick is to get the correct thickness of gasket so that the coverslip will hold the sample flat without squishing it.

I use the commercially available self-adhesive silicone gaskets that fit on traditional microscope slides. They come in 1 mm thickness, but they can be carefully stacked for thicker chambers. You then fill the chamber with the mounting solution, and top with a coverslip. As long as you are not using a permanent mounting solution, they are reusable. (I only use glycerol, agarose, or PBS as a mountant.)

If you have access to self-adhesive silicone sheets, you can cut your own. I have a friend who did that since she needed very deep chambers, cutting the holes with biopsy punches.

If your samples aren't very thick and you are using an aqueous mounting solution, you can also use Mat-Tek cell culture dishes. You put the sample in the culture well, and then carefully put a coverslip on top of the well. They are very helpful if you are using an inverted microscope system, but make sure to match the type of coverslip used by the manufacturer to your system optics, since you can order them with either #1 or # 1 1/2 coverslips.

Good Luck!

Jill

Hi Allesio,

I did some whole mounts on mammary glands of mouse. I mounted them with permount. Gently press the coverslip not with crushing force. It takes time to dry. If you are careful, you can take pictures under steromicroscope right away or let them dry for a day. The protocol and quality of picture is enclosed in a reprint. My tissues were very thick (may be 1mm or more). Probably your tissues are thinner, you will do better job.

Good luck,

Regards,

Subhash

Thank you for your replies!

I agree with Subhash C. Juneja 's method. I alternatively used ProlongGold/Diamond to mount the tissues. I used this method of whole mount for diaphragm, EDL, and soleus muscles in the past to look at NMJ distribution and nerve networks (see attached). Make sure to use enough mountant to cover the tissue and apply the weight until it is dry (otherwise the coverslip will pull off). For the EDL muscle, I cut it into a few strips, as it is too thick to mount completely for confocal. You should get good antibody penetration and suitable fields of view. See attached photos for examples. I have also attached a useful paper that details methods of staining I used to generate these images.

Thank you, Elliot for your feedback!

Whats the step to prepare a whole mount of the mesentery. Is there any link which can provide me the whole process of whole mount preparation of human mesentery

Thank you in advance

Chiman