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Table 2 Contrast stain formulations and protocols

From: MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues

Stain Stock solution Staining procedure
PTA 1% (w/v) phosphotungstic acid in water Mix 30 ml 1% PTA solution + 70 ml absolute ethanol to make 0.3% PTA in 70% ethanol. Keeps indefinitely.
   Take samples to 70% ethanol.
   Stain overnight or longer.
   Change to 70% ethanol. Staining is stable for months.
   Scan samples in 70% – 100% ethanol
IKI 1% iodine metal (I2) + 2% potassium iodide (KI) in water Dilute to 10% in water just before use.
   Rinse samples in water.
   Stain overnight.
   Wash in water.
   Can be scanned in water or dehydrated to alcohol.
I2E, I2M 1% iodine metal (I2) dissolved in 100% ethanol (I2E) or methanol (I2M) Use at full concentration or dilute in absolute alcohol.
   Take samples to 100% alcohol.
   Stain overnight or longer.
   Wash in alcohol.
   Stain does not need to be completely washed out before scanning.
Osmium tetroxide standard EM post-fixation Same as routine EM processing.
   Osmium-stained samples can be scanned in resin blocks, with some loss of contrast.
  1. Apart from osmium tetroxide, the reagents listed here are widely available, inexpensive, low-toxicity, and have been found to give good x-ray contrast to a wide variety of tissues. The stock solutions can be kept for months or longer at room temperature.