Iron stain kit prussian blue stain

Histological analysis was conducted to assess tissue iron accumulation using the Iron Stain Kit (Prussian Blue stain) (ab150674, Abcam, USA), following the manufacturer’s instructions. The iron in the tissue reacted with kit components, resulting in a blue color. Images were captured by a digital microscope and scanner (M8, Precipoint, Germany). Total iron was quantified by Iron Assay Kit (ab83366, Abcam, USA). Briefly, the lysed colon homogenate cell was added to 96-well plates for chemical reaction. Iron was released by reducing Fe³⁺ to Fe²⁺ by an acidic buffer. The released iron was then reacted with a chromagen and produced a colorimetric product (at 593 nm) proportional to the total iron level. Total iron concentrations were determined from the standard curve.

To verify the circulating level of Ly6G+, CD11b/c+ or myeloperoxidase (MPO) + cell populations, whole blood cells were stained and performed to flow cytometric analysis (FC500, Beckman) by day 28 after ICH induction. The 1.0 × 10⁶ cells were stained with Ly6g-Alexa Fluor 488 (Abcam), CD11b/c-PE (BD bioscience) or MPO-PE (Abcam) antibodies to detect cell surface markers.
In addition, to analyze circulatory levels of inflammatory biomarkers, serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 concentration were assessed by days 2 and 28 after ICH induction in duplicate with a commercial ELISA kits (R&D Systems).
Furthermore, prior to sacrificing the animals (i.e., by day 28 after ICH induction), the percentages of viable and apoptotic peripheral blood mononuclear cells (PBMNCs) were determined by flow cytometry using double staining with annexin V and propidium iodide (PI) which is a simple and popular method for the identification of apoptotic cells (i.e., early [annexin V+/PI−] and late [annexin V+/PI+] phases of apoptosis).
Finally, to investigate whether the Iron coated in the hUC-MSCs [i.e., ironic-magnetic-nanoparticles (Ir-MNa)- coated hUC-MSCs], the Iron Stain Kit (Prussian Blue Stain) (Abcam, ab150674) was utilized in the present. The procedure and protocol were according to the manufactory instructor.

Spleens were fixed in 4% paraformaldehyde in PBS for 24 h and then embedded in paraffin blocks. Paraffin sections of the spleen were prepared using a rotary microtome (RM2135, Leica, USA) and Prussian blue iron staining was performed. Iron Stain Kit (Prussian Blue Stain) (Abcam, ab150674) was used by following manufacturer’s procedure. Autofluorescence of RPM was observed using an IX70 inverted fluorescent microscope (Olympus, Tokyo, Japan).