Thorough Manual Paraffin Wax 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.
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.
Processing Schedule
- Hold tissues in NBF until the evening.
- 60% ethanol overnight.
- 80% ethanol until noon.
- 90% ethanol until evening.
- 95% ethanol overnight.
- First absolute ethanol until noon.
- Second absolute ethanol until evening.
- Third absolute ethanol overnight.
- Fourth absolute ethanol until noon.
- Fifth absolute ethanol until evening.
- First clearing agent overnight.
- Second clearing agent until noon.
- Third clearing agent until evening.
- Fourth clearing agent overnight.
- First wax at 58°C without vacuum until the evening.
- Second wax at 58°C under vacuum overnight.
- Third wax at 58°C under vacuum until noon.
- Fourth wax at 58°C under vacuum until late afternoon.
- Cast the blocks.
- Leave 48 hours at room temperature for the crystal structure to stabilize.
- Section.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.