Thorough 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.

Thorough processing
Tissue preparation
Thickness	About 5 mm thick. Increase times for thicker tissue and whole organs of fetuses or small animals.
Area	50 mm square, sometimes larger, determined by slide and coverslip area, and the capacity of the microtome used.
Fixation	Thorough fixation of bulk tissue ~ 1-2 weeks in 10% NBF. Breadloaf large organs.
Fixed Trimmed	24 hours minimum in 10% NBF, or thorough secondary fixation.
Unfixed Trimmed	48 hours minimum in 10% NBF, or thorough fixation in another fixative.
Reagent 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	Due to the length of time in clearing agent, this schedule should be used with chloroform. Since this poses health hazards, carry out the clearing steps under a fume hood. Cedarwood oil may also be used, but may require longer times for clearing.
Processing time	This schedule requires a minimum of 6 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. 60% ethanol overnight.
3. 80% ethanol until noon.
4. 90% ethanol until evening.
5. 95% ethanol overnight.
6. First absolute ethanol until noon.
7. Second absolute ethanol until evening.
8. Third absolute ethanol overnight.
9. Fourth absolute ethanol until noon.
10. Fifth absolute ethanol until evening.
11. First clearing agent overnight.
12. Second clearing agent until noon.
13. Third clearing agent until evening.
14. Fourth clearing agent overnight.
15. First wax at 58°C without vacuum until the evening.
16. Second wax at 58°C under vacuum overnight.
17. Third wax at 58°C under vacuum until noon.
18. Fourth wax at 58°C under vacuum until late afternoon.
19. Cast the blocks.
20. Leave 48 hours at room temperature for the crystal structure to stabilise.
21. Section.

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