Routine Manual Processing

In the schedules 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.

Routine processing
Tissue preparation
Thickness	About 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.
Times	All times in processing fluids for this schedule are minimums. Tissues may be left for much longer than the times specified and doing so does not harm them.
Clearing agent	Xylene can be used with this schedule, as can other clearing agents with somewhat longer times.
Processing time	This schedule requires 4 days from the time the tissues go into the first ethanol. Weekends should be timed so that the tissue is in an ethanol or chloroform, rather than wax.

 

1. Hold tissues in NBF until the evening.
2. 70% ethanol overnight.
3. 95% ethanol until noon.
4. First absolute ethanol until evening.
5. Second absolute ethanol overnight.
6. Third absolute ethanol until noon.
7. Fourth absolute ethanol until evening.
8. Fifth absolute ethanol overnight.
9. First clearing agent for 2 hours
10. Second clearing agent until noon.
11. Third clearing agent for 2 hours.
12. Fourth clearing agent until evening.
13. First wax at 58°C under vacuum overnight.
14. Second wax at 58°C under vacuum until noon.
15. Third wax at 58°C under vacuum until late afternoon.
16. Cast the blocks.
17. Leave at room temperature overnight for the crystal structure to stabilise.
18. Section.

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