Accustain iron deposition kit

The gastrocnemius muscle was excised from both hind limbs, dissected from fat and connective tissue, immediately fixed in Bouin’s solution for 24 h, and stored in 70% ethanol before further preparation. After dehydration, muscle was embedded in paraffin and cut into 7 μm sections with a microtome (Reichert-Jung, Germany) on the horizontal plane, transversely to the long axis of the muscle. The sections were placed on a slide and stained with haematoxylin and eosin. The morphology of muscle structure was studied by standard light microscopy (Olympus, type CH2) under × 2 and × 40 objectives.
Non-heme iron staining of the gastrocnemius muscle samples was analyzed using Accustain Iron Deposition Kit (Sigma–Aldrich). After mounting on glass slides, muscle sections were deparaffinized, incubated with working solution containing Perls’ Prussian blue for 30 min, counterstained with pararoseaniline solution for 2 min and analyzed under standard light microscopy (Olympus, type CH2).

Non-heme iron deposits were analyzed using the Accustain Iron Deposition Kit (Sigma Aldrich). Briefly, duodenal samples excised immediately after sacrifice were fixed in Bouin’s solution for 24 h, then stored in 70% ethanol. After embedding in paraffin, the samples were cut into 7-μm sections using a microtome (Reichert-Jung, Germany). The sections were placed on a slide, deparaffinized and incubated with Perls’ Prussian Blue solution for 30 minutes. Slides were counterstained with pararoseaniline solution for 2 minutes and examined with a standard compound light microscope (Olympus, type CH2).

Non-heme iron deposits were analysed using Accustain Iron Deposition Kit (Sigma Aldrich). Briefly, liver samples were excised immediately after sacrifice and fixed in Bouin’s solution for 24h, then stored in 70% ethanol. After embedding in paraffin, the samples were cut into 7-μm sections with a microtome (Reichert-Jung, Germany). The sections were placed on a slide, deparaffinised and hydrated tissues were incubated with working solution containing Perls’ Prussian Blue for 30 min. Slide were counterstained with pararoseaniline solution for 2 min and analysed under standard light microscopy (Olympus, type CH2).