Weigerts hematoxylin

Tissues were fixed for 24 h in 10% neutral buffered formalin (Sigma), dehydrated in ethanol gradients, paraffin embedded and sectioned at 3 μm on polylysine slides (Thermo scientific). Slides were rehydrated with decreasing percentage of ethanol solutions and subjected to stains.
For iron staining, slides were quenched with 3% H₂O₂ and treated for 20 min with Prussian Blue (Sigma Aldrich) followed by a 20 min incubation with 3,3′-diaminobenzidine (DAB) (Sigma Aldrich). Meyer's hematoxylin was used as counterstain.
For Masson's trichrome staining, slides were treated overnight with the Bourin's mordent solution (Roth). After washing, slides were treated for 5 min with Weigert's hematoxylin (Roth), 5 min with Biebrich Scarlet-Acid Fuchsine (Sigma Aldrich), 5 min with Phosphotungstic/Phosphomolybdic Acid Solution (Sigma Aldrich), 5 min with Aniline Solution (Sigma Aldrich) and 2 min with 1% acetic acid (Carl Roth).
For hematoxylin and eosin staining, slides were treated for 6 min with Mayer's Hematoxylin (Sigma Aldrich), rinsed in phosphate buffered saline, and incubated for 2 min in Eosin in acetic acid (Sigma Aldrich).
After the stains, slides were dehydrated with ethanol gradients and mounted with Entellan (Sigma Aldrich). Digital images were acquired up to 40 × magnification with an automated slide scanner (Aperio - Leica biosystems)