Epr6454

We randomly selected 3 patients with Crohn’s disease, 3 patients with ulcerative colitis, and 1 patient with diverticulitis (with normal colonic mucosa) who underwent surgical resection at Gangnam Severance Hospital between January 2022 and January 2023. We reviewed all hematoxylin and eosin (H&E) slides used at the time of diagnosis. We selected regions with creeping fat in Crohn’s disease, mucosal ulceration in ulcerative colitis, and normal colonic mucosa in diverticulitis for Masson’s trichrome and immunohistochemistry by light microscopy. Masson’s trichrome and IHC staining were performed on 4 μm sections obtained from selected formalin-fixed paraffin-embedded (FFPE) blocks. The IHC staining for PTX3 (1:1000, sc-373951, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and CD138 (1:1000, EPR6454, rabbit monoclonal, Abcam, Cambridge, UK) was performed using Benchmark^® automatic immunostaining device (Roche Tissue Diagnostics, Tucson, USA) and an UltraViewTM Universal DAB Detection Kit (Ventana Medical Systems, Tucson, USA), according to the manufacturer’s instructions.

The sections were blocked by 20% Block Ace (Dainippon Sumitomo Pharma Co., Osaka, Japan) in 0.1 M PB with 0.005% saponin (Sigma-Aldrich Co., St. Louis, MO) and incubated with the primary antibody, rabbit monoclonal anti-syndecan-1 antibody (EPR6454; abcam), followed by the secondary antibody, goat anti-rabbit IgG antibody (Goat Anti-Rabbit IgG H&L [HRP]; abcam), overnight at 4 °C. The antibodies were dissolved in 0.1 M PB containing 5% Block Ace and 0.005% saponin. The sections were washed with PBS after each reaction. Finally, they were mounted in 0.1 M PB-glycerin (1:1) solution and observed under a fluorescent microscope (BZ-9000; Keyence Co.). For negative control, nonimmune serum was substituted for the primary antibody.
To investigate the distribution and engrafting of transplanted BMMNCs in the host tissues, we also injected GFP-derived BMMNCs into the heatstroke rats (GFP-BMMNCs group, n = 9). Sections of lung, kidney, and spleen were observed under a fluorescent microscope (BZ-9000; Keyence Co.) at 1 day (n = 3), 1 week (n = 3), and 2 weeks (n = 3) after heat stress.

SDC1 was localized by immunohistochemistry in placental tissue collected from either fetal growth restricted or preterm control pregnancies. In brief, paraffin sections (5 µm) were dewaxed in xylene and rehydrated through descending grades of ethanol. Sections underwent antigen retrieval via microwaving using 0.01 mol/l sodium citrate buffer (pH 6.0) for 20 min and then incubated in the hot buffer for a further 20 min. Sections were washed for 10 min in Phosphate-buffered saline pH 7.6 (PBS). Following endogenous peroxidase quenching and blocking of non-specific binding, sections were incubated at 37 °C for 1 h with anti-SDC1 antibody (EPR6454, Abcam, Cambridge, UK) in blocking buffer (DAKO). For isotype controls, primary antibody was substituted with rabbit IgG (SC2027, Santa Cruz, Texas, USA). Staining was visualized using the HRP/DAB Detection IHC Kit (Abcam, Cambridge, UK), and lightly counterstained with Harris hematoxylin (Accustain). Sections were then dehydrated and mounted. Staining was visualized and captured using a Leica microscope and camera.