Hi all,

I'm trying to utilize the Perls Prussian Blue staining method to look at iron overload in fixed bone tissue (specifically the bone matrix, around osteocytes) and, so far, have had little luck.

I'm using a protocol adapted from this kit from Newcomer Supply (https://www.newcomersupply.com/product/iron-gomori-prussian-blue-stain-kit/) and have used both my own reagents and the solutions provided by the kit (in separate attempts). My samples are of mouse tibiae, fixed in 10% NBF for 48 hours, decalcified in 0.5M EDTA for 2 weeks, embedded in paraffin, cut at 5 microns. Either xylene or CitriSolv is used as a deparaffinizing agent. See attached protocol for specific steps. I am able to see some very light blue specks in the bone matrix, but they're seemingly random in size, intensity, and location of appearance- and are not within the osteocyte lacunae.

Would anyone be able to either provide reasoning as to why this type of stain would not work well for detecting iron around osteocytes (or other bone cells)? Alternatively, does anyone have suggestions for detecting iron accumulation in bone (in vivo/ex vivo)?

Any experienced insight to this would be helpful, thank you!