Triple Staining Technique for Histology

A triple staining with Aniline blue, acid Fuchsin and Orange-G (AFOG) was performed as previously described [7 (link)]. For the single-dye staining with Aniline blue, cryosections were refixed with 10% neutral buffered formalin solution for 15 min and washed in PBST for 10 min. The slides were transferred into preheated Bouins fixative (Lot. 1536576, Reactorlab) and incubated for 2.5 hours at 56°C and another hour at room temperature. The slides were washed twice for 20 minutes in tap water and then incubated in 1% phosphomolybdic acid for 5 minutes. After rinsing with distilled water, sections were incubated for 4 minutes with aniline blue staining solution (1 g aniline blue diammonium salt, Sigma Aldrich, 415049; 200 ml acidified distilled water) and again washed with distilled water. The sections were dehydrated in 95% ethanol and twice in 100% ethanol for 2 minutes. After passing through three consecutive xylol baths, the slides were mounted with Entellan (107961, Merck Millipore).