Dear Yong,

I can understand what your problem is. Because you have the epithelial layers (lining of the organ of corti, hair cells, Reissners' membrane etc) just holding on to the bone/cartilage they easily peal off and wash away.  Therefore it is difficult to do the same methodology as in brain sections FFPE. I work with rat cochlea but use a slighly different method which works well. I have been working with rat cochlea and inner ear slices without the paraffin. I basically fix my tissues in PFA, and then freeze the entire cochlea. Then I cut 20-50 micron thick sections and do IHC on floating sections. For details of procedures and references see my publications listed below.

Best wishes,

Refik

Article Postnatal developmental expression of the PDZ scaffolds Na+/...

Did you cut other tissues with success, that were processed equally? time in formalin, alcohols, xylen, paraffin?

To my mind comes the picture of underprocessed fat, that breaks or even explodes on the water-surface. Here it would help to increase the time in xylen and paraffin.

You can also try to let the slides float on a cold water, mount on an adhesive glass-slide and lay the glass-slide on the warm margin of the waterbath. This could lead to some folds, but holds the part of the tisse in place.

Dear Yong,

Cut 5 micron size thickness and float on warm water bath, use small brush to lift the sections instead of needle. Use APS coated glass slide to mount sections.

Here is an alternate method of sectioning:  Use positive charged slides (plus slides or silaned slides) that have a small drop of glycerol smeared on them.  After sectioning the number of sections you want, place them directly on the slide.  Slowly add deionized water with a pipet so that the water goes beneath the sections - just enough to cover the slide.  Place the slice on a hot plate or slide warmer that is about 50-55oC.  Watch as the section or ribbon expands.  Carefully drain off excess water and place on a slide warmer set at about 40-44oC to dry overnight.  Once you get use to this method, it isn't too difficult and you loose less tissue.  I use it when I am doing serial sectioning.