Overnight Automatic Processing

In the schedule below, it is presumed that the working day is from 8:00 a.m. to 5:00 p.m.. If other than that, appropriate adjustments should be made.

Overnight processing
Tissue preparation
Thickness	No more than 3 mm thick.
Area	20 mm × 30 mm.
Fixed tissue	Breadloaf large organs and store in NBF for 48 hours. Select tissue from fixed areas, trim to size and refix until the evening. If the trimmed sample is visibly unfixed, refix for a further 24 hours.
Unfixed tissue	Slices of tissue should be thoroughly fixed before processing. Fix at least 24 hours after trimming to size. Biopsies may be described and wrapped then submitted. The treatment with NBF at 65°C should be adequate for minimal fixation.
Times	All times in processing fluids for this schedule are for tissues 3 mm thick or less. Tissues thicker than that will require longer times.
Clearing agent	Xylene or another clearing agent that will clear tissues in similar times should be used.
Processing time	This schedule processes overnight. On weekends tissues should be left in fixative until Sunday evening with a 48 hour delay (on a carousel processor) or a weekend extended processing schedule established (on a fluid transfer processor).

 

It has been presumed that the processor has 12 stations. If it has more than 12, increase the number of absolute ethanol stations first as clearing and wax infiltration proceed faster with complete dehydration. If it is possible to elevate temperatures, do so in all stations.

1. Trim fixed tissues during the day and keep in NBF until 4.30 p.m..
   Place in an oven at 65°C for 2 hours during this time.
   Place on processor.
2. NBF 2 hours with elevated temperature but not to exceed 65°C.
3. 70% ethanol 1 hour with elevated temperature.
4. 95% ethanol 1 hour with elevated temperature.
5. First absolute ethanol 1 hour with elevated temperature.
6. Second absolute ethanol 1½ hours with elevated temperature.
7. Third absolute ethanol 1½ hours with elevated temperature.
8. Fourth absolute ethanol 2 hour with elevated temperature.
9. First clearing agent 1 hour with elevated temperature.
10. Second clearing agent 1 hour with elevated temperature.
11. Third clearing agent 1 hour with elevated temperature.
12. First wax at 58°C under vacuum 2 hours.
13. Second wax at 58°C under vacuum 1 hour.
14. Third wax at 58°C under vacuum 1 hour.
15. Cast the blocks, allow to skin over, then gently lower into cold tap water until solid.
16. Trim and face blocks, then store face down on ice until sectioned.

Step 14, the third wax bath, is done off the processor.

It should be understood that this processing schedules is a guide only, and the times given may need to be altered to take the type of tissues being processed into account, as well as other local conditions.

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