Anti chromogranin a

Immunohistochemistry was performed on 4.5 µm sections. Sections were de-paraffinized and rehydrated by passing through xylene and a series of ethanols to water. Antigen retrieval was performed by boiling in 10 mM citrate buffer (pH 6.0) for 10 minutes. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 30 minutes. Sections were then subject to rabbit VECTASTAIN Elite ABC horseradish peroxidase kit following the manufacturers protocols. Sections were developed in DAB for 5 minutes, counterstained in hematoxylin, dehydrated with ethanol, cleared in xylene and mounted in DPX. Primary antibodies; Anti-Ki67 (Thermo Scientific, Fremont, CA, USA; clone: SP6; 1:200), Anti-p-H3 (Merck Millipore, Germany; 06–570: Anti-phospho-Histone H3 (Ser10) Antibody; 1:500), Anti-lysozyme (Life Technologies; A0099), Anti- Chromogranin A (Abcam; ab15160), Anti-CD11b antibody (Abcam; ab133357; 1:4000), Anti-CD3 (Themo Scientific, RM-9107-R7; prediluted), Anti-CD45R (Thermo Scientific, RA3-6B2, 1:100). Alcian blue staining was performed following standard procedures in 1% Alcian blue in 3% acetic acid, pH 2.5.

The paraffin-embedded sections of ileum and colon tissue were prepared and processed for HE as described previously (11 (link)). The ileum and colon tissues pathology of the proband in HE was examined by light microscope (LM) (H500S, Nikon). Immunohistochemical staining of the paraffin-embedded sections was performed according to EnVision system protocol. The sections were incubated with anti-Chromogranin A (1:200, Abcam) and with HRP-labeled secondary antibody (DAKO). The localization and distribution of intestinal endocrine cells were examined under the H500S LM, and the images were processed by Image-pro Plus software, version 9.3 (Media Cybernetics).

The small and large intestines of house musk shrews were both resected 1–2 h after oral administration of the spores and fixed using the Swiss roll method [19 (link)]. Paraffin blocks were sectioned at 5–6 μm thickness and deparaffinized. For immunohistochemical staining, ribbons were rehydrated, with endogenous peroxidases being quenched with 3% H₂O₂ in methanol for 30 min in the dark. After washing with PBS for 20 min, the ribbons were blocked with 1% bovine serum albumin (Sigma–Aldrich, St. Louis, MO, USA) for 20 min. The ribbons were then incubated with primary antibodies at the following dilutions in blocking buffer: Mouse anti-serotonin (1:250, LSBio, Seattle, WA, USA), anti-c-fos (1:100, Abcam, Cambridge, UK), anti-mast cell tryptase (1:200, Abcam), anti-chromogranin A (1:400, Abcam), and anti-CaMKI (1:50, Abcam). After washing in PBS, the ribbons were incubated with the secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG (1:500, Cell Signaling, Danvers, MA, USA). After washing, the color was developed using a substrate, DAB (3,3′-diaminobenzidine tetrahydrochloride), before being counter-stained with hematoxylin.