[Histonet] Human bone and soft tissue processing issues

[M.O.]

 Hello Histonet!



I am hoping that someone could shed some light on some issues I am having
with some human knee tissues embedded in paraffin.  About one year ago, we
started having some issues with the established protocol and decided to
increase the processing times.



Issues we are seeing:

Once processed, the stained ligaments (MCL, LCL, ACL, etc) appear to have
brittle tissue (sort of wavy looking) toward the middle of the sample in
stained sections.  The block itself has white brittle areas that flake off.



Regarding bone slabs, we find that the junction between the cartilage and
bone is where the tissues are the hardest after decal and the color is
different in this area.  We think that the fixation may be a problem
because of penetration.  If you have any suggestions for ensuring complete
fixation, that would be very helpful!  The processor run does not include a
fixative station.



Soft Tissues and Ligaments:

All tissues are trimmed down to about 4mm in thickness and put into
Anatech’s Z-fix for 5 days with one change of Z-fix. Washed and put into
70% EtOH.



Bone:

Slabs are 6-9mm thick (we ideally aim for 7mm) across the tibial plateau
and along the medial and lateral femoral condyles – with cartilage and
about 1cm of subchondral bone.  The slabs are individually fixed with Z-fix
for 1 week, with one change of Z-fix and excess bone trimmed after the
first 3 days.



Then the samples are put into TBD-2 (formic acid decal) for up to 2 weeks
depending on the end point, with a change of decal.  Once decalcified, the
samples are cut into 20mm X 4mm X 7mm pieces and washed and put into 70%
EtOH.



The processor completes all steps under vacuum.



Older Protocol with another processor:

70%, 80%, 2X95%, 3X100%, 3XPropar, 3XParaffin

All reagents are 2 hours.



Old Protocol with Thermo Scientific Excelsior Processor:

70%, 80%, 2X95%, 3X100%, 3XPropar, 3XParaffin

All reagents are 4 hours.



Recent Protocol (past year) Thermo Scientific Excelsior Processor:

70% 1hr

85% - 4hrs

95% - 4hrs

2X100% - 4hrs

100% - 5hrs

2XPropar - 4hrs

Propar - 5hrs

2XParaffin - 4hrs

Paraffin - 5hrs





The main questions are:

1)   How can we ensure complete fixation?

2)   What is causing these brittle/hard areas in our tissues, even for soft
tissues?

3)   What does over-processing look like in samples?

4)   Is our recent protocol okay or does it need adjustments?



Thank you in advance for any help or suggestions!



Sincerely,

Merissa