Ba400digital

The streptavidin-peroxidase (SP) method was performed in strict accordance with the kit instructions. 4 μm thick liver tissue sections were routinely dewaxed and hydrated with gradient ethanol. The samples were treated with antigen repair solution at 95–99 ℃ for 40 min and cooled at room temperature for 20 min. After washing 3 times, Ki-67 primary antibody (No. ab15580, Abcam, Cambridge, MA, USA; 1:100) or CK19 primary antibody (No. bs-15590R, Bioss, Beijing, China; 1:100) was added and incubated overnight at 4 ℃. Then the EnVision detection and color development kit was used for DAB color development, hematoxylin re-staining, gradient ethanol dehydration, xylene transparency, and then treacle sealing for observation. IHC images were evaluated microscopically (BA400Digital, Motic Instruments, Inc., Baltimore, MD, USA).

At the end of the animal study, goats were sacrificed for sampling. Then, the rumen and small intestine were collected and fixed with 4% formaldehyde, rooted in paraffin, and marked with eosin and hematoxylin, as previously reported [11 (link)]. Images of small intestine samples were observed under electron microscopy (BA400Digital; Motic China Group Co., LTD., Xiamen, Fujian, China). Papillae length, papillae width, epithelium thickness of rumen, villus length, crypt depth, and villus width of the small intestine were evaluated with Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Ileum and colon tissues were divided into 3 μm sections through paraformaldehyde and paraffin embedding. Next, these samples were stained using haematoxylin and eosin (H&E) to assess the histological morphology. The main procedures included dehydration, pruning, embedding, sectioning, staining, and sealing. Tissue sections were observed and collected by a digital three-eye camera system (BA400 Digital, MOTIC China Group Co., Ltd. Xiamen, China).

The abundance of SIgA was assessed for paraffin-embedded slides after the sections were dewaxed. Endogen biotin and non-specific signals were blocked with appropriated reagents. Antigen retrieval was carried out in a microwave oven (two cycles for 5 min each at 780 W, in citrate buffer, pH 6.0, twice washed in PBS for 5 min each). Then, the treated slides were overnight incubated with primary antibodies at −4°C in a humidified chamber, washed in PBS, and visualized by biotinylated secondary antibodies followed by staining by DAB kit for 2 min and washed in distilled water, and finally counterstained with hematoxylin, dehydrated, transparentized, and sealed. At least 10 fields of view from each sample by BA400 Digital (Motic China Group CO., LTD.) were analyzed with the imaging system for each protein of interest. All tissues were observed under 100 times of each slice, and then, three visual fields were selected to collect 400 times of microscopic images, respectively. The integrated optical density (IOD) and area of all collected images are measured by Image pro Plus 6.0, and the mean density of each image is calculated. The average optical density of three images is used to calculate the average, and the average optical density of each sample is obtained.