[Histonet] Processor Troubleshooting

[Jayasri Narasimhan]

Hi everyone,

For the past two months in the research lab I work in, we've been dealing with poorly processed mouse livers. The following is the protocol, which is optimized for mouse mammary tissue, but we've been using it for all tissues.

Fixation:
~48 hours in 10% nbf, shaking.
70% ethanol until processing; it sits in there anywhere from a couple of days to weeks.

Processing (Sakura Tissue Tek VIP - 5)
70% ethanol       5 min
80%                        30 min
95% - 1                  30 min
95% - 2                  40 min
100% -1                30 min
100% -2                30 min
100%-2                 30 min
Xylene-1              45 min
Xylene-2              40 min
Xylene-3              30 min
Paraffin1              40 min
Paraffin2              45 min
Paraffin3              45 min

The worst livers expanded on the ice bath, and had the hepatocytes appeared to have perinuclear cytoplasmic clearing. We ran PAS and glycogen doesn't fully account for their appearance.

We've pm'ed our processor, compared runs on our processor to runs from the research histocore, and processed a few cassettes in surgical pathology using their protocol.

By far, livers processed in Surg Path were the best. Their protocol starts with NBF and then goes into 80% alchohol. From then, each cycle is 40-60 minutes, so a bit longer than ours.

Finally, the Sakura tech ran a warm water flush. The specimens run after this flush look similar to the ones run at the research histocore, so the processing problems seem to be resolved.

My main question: what could the warm flush have done to resolve our processing issues? Since we're storing our tissues in 70% before putting them on the processor, aren't we preventing salt precipitation that could affect processing? Does anyone know the chemistry/science behind what might have happened here?

Thanks,
JN
OHSU Lab