Hematoxylin solution

In addition to H&E staining, immunohistochemistry was performed on 4-µm-thick formalin-fixed paraffin-embedded tissue sections. Briefly, after dewaxing and rehydration at 60 °C, the sections were treated with citrate buffer in a microwave oven for 30 min to retrieve nuclear antigens. The tissue sections were then blocked for endogenous peroxidase activity and incubated overnight at 4 °C with antibodies. The recommended dilution ratio was used for all of the antibodies. Secondary antibodies were added for an hour at room temperature (Maxim, China), and antibody detection was revealed by the DAB substrate (Vector Laboratories, USA), followed by costaining with hematoxylin solution (Sangon, China). The results and previous H&E staining slides were reviewed and evaluated at low magnification by two expert pathologists. The staining was scored as 4+ (positive in greater than 50% of cells), 3+ (positive in 25–50% of cells), 2+ (positive in 5–25% of cells), 1+ (only weak reactivity or less than 5% of positive cells), or 0 (no reactivity).

The intestinal tissues were fixed in 10% neutral formalin, followed by dehydration in ascending series of ethanol, transparention using xylene and paraffin embedding. Subsequently the paraffin-embedded tissues were cut into 5 μm-thick sections, dewaxed in xylene, and rehydrated through decreasing concentrations of ethanol. The slides were successively incubated with hematoxylin solution (Sangon Biotech, Shanghai, China) for 10 min and eosin solution (Sangon Biotech, Shanghai, China) for 3 min, followed by dehydration with graded ethanol. After the addition of xylene, the sections were observed using a light microscope (Nikon, Tokyo, Japan).
Pathologic scoring was performed using hematoxylin and eosin (H & E) staining data in a blinded manner. Three independent parameters of each sample were examined, including severity of inflammation (0–3: none, slight, moderate and severe), depth of injury (0–3: none, mucosal, mucosal and submucosal, and transmural) and extent of crypt damage (0–4: none, basal 1/3 damaged, basal 2/3 damaged, only surface epithelium intact, and entire crypt and epithelium lost).^{23 (link)} The sum of these parameter scores was multiplied by a factor which reflected the percentage of tissue involvement (1–4: 0%-25%, 26%-50%, 51%-75%, and 76%-100%).