After Puchtler et al. 1962

*This protocol is appropriate for tissue, but does not work on hagfish slime threads.

• Fix in 4% paraformaldehyde in PBS
• Rinse 3x in dH2O
• Stain 10 minutes in Mayer’s acid hemalum
• Rinse 3x in dH2O
• Immerse in alkaline pretreatment working solution for 20 minutes
• Stain in Congo red working solution for 20 minutes, rinse thoroughly in dH2O
• Dehydrate rapidly in 3 changes of absolute ethanol
• Clear in xylol, mount in Permount

Stock Solutions
a. 80% EtOH saturated with NaCl
b. 80% EtOH saturated with Congo Red and NaCl
c. 1% NaOH

Working Solutions
a. Add 50 μl 1% NaOH to 5 mL stock solution a, use within 15 minutes
b. Add 50 μl 1% NaOH to 5 mL stock solution b, filter, use within 15 minutes

Stock solutions are good for a few months
About 1.1 g of NaCl is enough to saturate 50 mL of 80% EtOH
A 0.5% CR solution is more than saturated