Iron Stain

The Specimen Collection section of the Instructions For Use states: "Any well prepared tissue section cut 5–6 microns and fixed in neutral buffered formalin is satisfactory for iron staining. Blood or bone marrow films are prepared in the usual fashion, air-dried at least 30 minutes and fixed in absolute methanol for 7 minutes."

If your free floating sections are fixed in PFA, this is really the same as fixing in Neutral Buffered Formalin. Once the bonds of the paraformaldehyde are broken, the active molecule is formaldehyde/formalin. Once the free floating sections have been fixed and rinse in water, the free floating sections can be stained with the HT20 procedure.

This kit includes 250 mL each of a 4% Potassium ferrocyanide solution and 1.2 mM Hydrochloric Acid solution and 30 mL of 1% pararosaniline soluition in methanol. These reagents are used to prepare working solutions that must be used once time and discarded. The number of tests per kit depends on the format and/or number of slides. For example, the working iron stain solution is prepared from equal parts potassium ferrocyanide solution and hydrochloric acid solution. For example, if 40 mL of working solution is prepared, this kit will be sufficient for 12 staining events. Please see the link below to review the product datsheet for additional details:
https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/321/539/procedure-ht20-ironstain-ms.pdf

Both frozen sections and paraffin-embedded tissue are considered satisfactory for iron staining, as per the Instructions For Use. Blood or bone marrow films are prepared in the usual fashion, air-dried for at least 30 minutes, and fixed in absolute methanol for 7 minutes.

While slides anticoagulated with EDTA may be suitable for demonstrating iron in peripheral blood smears and bone marrow smears, bone marrow that has been decalcified in EDTA may not demonstrate iron using the Perl’s or Prussian Blue Method. Even for bone marrow smears, EDTA can diminish or remove iron from bone marrow aspirates if the levels of EDTA used in collection are high. EDTA has various uses in laboratories, including as an anticoagulant and a decalcifying agent to chelate calcium from bone. By removing the calcium, bone can be processed into paraffin blocks and cut into thin sections using a microtome. EDTA can also be used to chelate a number of other metals, including iron. Once the iron deposits have been removed from osseous bone, the iron deposits are also removed from cells at the same time. Consequently, bone marrow smears that have been decalcified in EDTA will generally stain negative for iron in bone marrow slides.

It's uncertain whether it would be possible to dual stain a tissue section with a Prussian Blue staining procedure and the IHC kit ECM590. However, it's recommended to try the ECM590 kit and then perform the Prussian Blue staining procedure. The color contrast should not be a problem, but the use of DAB for demonstrating both anti-von Willebrand Factor and anti-CD31 primary antibodies may lead to brown staining for both antibodies.

Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product.

Blood and bone marrow samples should be drawn without the use of EDTA as an anticoagulant. The EDTA chelates enough Iron to interfere with adequate staining. We advise using heparinized blood or freshly drawn blood which has not been anticoagulated.

Iron in tissue samples (used for research) can be visualized without the Pararosaniline counterstain, which is used to allow improved visualization of the tissue.

This kit can be used to stain cultured mammalian cells, which should be treated as blood or bone marrow and follow instructions given for specimen collection in the Package Insert.  We advise using some sort of positive control.

The reagents included in this kit are not available for separate purchase.