Wiegert s iron haematoxylin solution

Whole depth rumen wall tissue and enzymatically separated epithelium and LP were embedded in Tissue-Tek® O.C.T compound (Sakura, CA, USA), snap frozen in liquid nitrogen and cryostat (cryotome SME, Life sciences, Cheshire, UK) sectioned (10 μm). Sections were stained in Wiegert’s iron haematoxylin solution (Sigma, MO, USA) for 6–8 min, and then rinsed in tap water to clear unbound stain. Sections were then immersed in Gomori trichrome stain (Sigma, MO, USA) for 10 min, and the colour differentiated with 0.2% acetic acid. Sections were then dehydrated in ascending ethanol 95–100% for 2 × 5 min each change of solution. The sections were then cleared with xylene substitute (Sigma, MO, USA) for 3 × 5 min. A coverslip was mounted with DPX solution (Sigma, MO, USA), and air dried overnight.

Sample sections were prepared as described in HE staining. Cell nucleuses were stained by Wiegert’s iron haematoxylin solution (Sigma-Aldrich) for 10 min. Following thoroughly rinsing with PBS for 3 times, the tissue sections were dyed by Masson-Ponceau-acid fuchsin solution (0.7%, Sigma-Aldrich) for around 10 min. Sections were then rinsed in 2% glacial acetic acid and differentiated with phosphomolybdic acid for 4 min, followed by the staining with 2% aniline blue dye reagent (Sigma-Aldrich). The sections were imaged using a light microscope (Olympus, Tokyo, Japan) after dehydrating by ethanol series.

Mice cardiac tissues were fixed with 10% formaldehyde for 24 hours at room temperature, then decalcified, dehydrated, permeabilized with xylene, embedded in wax and finally sliced into 5‐μm‐thick sections with a microtome. Wiegert's iron haematoxylin solution (Sigma‐Aldrich, St. Louis, MO, USA) was used to dye the cell nucleus for 5 minutes. Following rinsing with distilled water 3 times, the sections were stained with 0.7% Masson‐Ponceau‐acid fuchsin staining solution (Sigma‐Aldrich) for 10 minutes. Samples were then rinsed in 2% glacial acetic acid and differentiated in phosphomolybdic acid for 4 minutes. The sections were directly stained with 2% aniline blue dye solution (Sigma‐Aldrich). Following dehydrating with ethanol series, clearing with xylene and mounting with neutral resins, images of the stained sections were captured with a light microscope.