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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.