u/CalligrapherHuge6821

[Reposted to include H&E staining for better visualization]

Pictured: murine lumbar spine H&E

I'm currently working as an RA in an academic lab and need to get better at longitudinal bones sectioning. Previously bone marrow was crackling too much, but it seems to be a bit better as long as the paraffin block is chilled a bit before sectioning. However, trabecular bone looks like its peeling away while the gap left behind maintains the correct shape. Cortical bone looks like its wants to wrinkle, too.

Really hoping this is a fixable, technique issue and not a processing thing. Any advice welcome!!

- fixed 48hr in PFA prior to decal

- 12 day decal in EDTA, confirmed with Xray

- 5-7um sections

- 6 degree clearance angle

- ice water-soaked gauze and 10% formic acid swab pressed against block between sections

- 40C degree water bath for ~10sec before scooping onto slide

- placed on slide warmer until water dries out

- 55C orienter is available to briefly melt paraffin and unwrinkle samples, but it seems to cause more problems with maintaining morphology than it fixes

I cut longitudinal sections of decalcified mouse bones for my research lab and I am struggling to get consistently good slides. Previously had issues with bone marrow ripping that was fixed by pressing an ice water soaked gauze against the block before sectioning, but now the trabecular bone looks like its shrinking away. I am smat a loss and my PI is breathing down my neck about getting it figured out. Photos are from scope after sections have dried out. I'm really praying this is a fixable sectioning technique issue and not an issue with processing:

-5um-7um sections

-ice water soaked gauze+ 10% formic acid pressed to tissue between sections to solidify paraffin and fix tissue

-6% clearance angle

-40C water bath with gelatin to unwrinkle ribbons, scooped to slide after about 15sec

-sits on a 42C slide warmer until water dries out