Prussian blue reaction

We used an iron staining kit based on the Prussian blue reaction (ScyTek Laboratories Logan, UT, USA) to identify iron accumulation in lung tissues [14 (link)]. Briefly, following deparaffinization and rehydration, 5-µm lung sections were counterstained with nuclear rapid red solution, dehydrated with alcohol, cleaned with xylene, and mounted on cover plates for examination. Ferric iron is colored blue in this stain, while the nuclei are colored red. Iron deposition was quantified by counting the positive Prussian blue stained cells in randomly selected 50 high power fields (magnification 400×)/group, which were taken and uploaded to a computer screen for analysis using the Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). For final calculations, data generated from all sampled sections, fields, and counts were used.

An iron staining kit based on the Prussian blue reaction (ScyTek Laboratories, Logan, UT, USA) was used to detect iron deposition in lung tissues. After deparaffinization and rehydration, the 5-µm lung sections were treated with equal volumes of 2% hydrochloric acid solution and potassium ferrocyanide solution for 5 min. The sections were then counterstained with nuclear fast red solution for 5 min, dehydrated with alcohol, cleared with xylene, and mounted with coverslips for further observation. This staining is such that ferric iron shows up as blue and nuclei as red. Positive cells were scored in 4 fields randomly selected from each section using a light microscope and the results were expressed as positive staining cells per high-power field (magnification 400×).