Rabbit anti sox10

Immunohistochemical staining analysis was performed as previously reported [5 (link), 14 (link)]. Briefly, the samples were fixed with 3.7% formaldehyde (FUJIFILM Wako Pure Chemical Industries) and permeabilized with PBS containing 0.2% Tween-20 (FUJIFILM Wako Pure Chemical Industries). Samples were then blocked with PBS containing 4% Block Ace (DS Pharma Biomedical) and 0.2% Tween-20 and then incubated with primary antibodies (mouse anti-NGFR (1:200; Advanced Targeting System) and rabbit anti-SOX10 (1:500; Abcam)) overnight at 4°C. Samples were then washed with PBS containing 0.2% Tween-20 thrice and then incubated with secondary antibodies (anti-mouse Alexa Fluor-488 and anti-rabbit Alexa Fluor-555; 1:1000; Thermo Fisher Scientific). For nuclear staining, 0.2 μg/ml Hoechst 33342 (Dojindo Molecular Technologies) was added to the samples. The samples were placed on the stage of the inverted microscope (IX81; Olympus), and fluorescence was observed with an electron multiplying charge-coupled device camera (iXon; Andor). The observed images were analyzed using ImageJ software (National Institutes of Health; available at http://imagej.nih.gov/ij/).

Intestinal tissues were collected and fixed overnight in 4% paraformaldehyde. The fixed intestinal tissues were OCT-embedded, snap-frozen, and prepared into 16 μm sections for immunofluorescence staining. Cell samples including the primary EGCs, primary astrocytes and Caco-2 cells were seeded in 24-well plates plated with cell climbing slices respectively. After stimulation, cell samples were fixed in 4% paraformaldehyde and washed thrice with PBS for immunofluorescence staining. After permeabilization and blocking, the tissue sections or cell samples were incubated with the respective primary antibodies overnight at 4 °C. Briefly, the primary antibodies consisted of chicken anti-GFAP (1:200, GeneTex, Irvine, CA, USA), rabbit anti-Iba-1 (1:200 Abcam, Cambridge, UK), rabbit anti-Sox10 (1:500, Abcam, Cambridge, UK), anti-CD40 (1:200, Abcam, Cambridge, UK), and ZO-1 (1:200, Abcam, Cambridge, UK) antibodies. Next, the tissue sections or cell samples were washed thrice with PBS and incubated with A488 anti-chicken (1:1000, GeneTex) or A594 anti-rabbit (1:1000, GeneTex) antibodies at 37 °C for 2 h. DAPI (1:1000, Invitrogen) was used for nuclei counterstaining. An Olympus FluoView FV1000 microscope (Olympus, Tokyo, Japan) was used to collect the fluorescent images of intestinal tissues and cell samples. Fluorescent intensity was analyzed by ImageJ.

In order to evaluate the biological properties of hDPSCs seeded on titanium and titanium–zirconia (CERID) disks before and after treatment, stemness markers were analyzed. Particularly, after 72 h of culture, cells were fixed and permeabilized as described above. After blocking with 3% bovine serum albumin (BSA) in PBS, cells were immunolabeled with mouse anti-nestin (Merck Millipore, Burlington, MA, United States) and rabbit anti-SOX10 (Abcam, Cambridge, UK) primary antibodies and subsequently targeted by AlexaFluor-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MS, USA) [26 (link)]. A Nikon A1 confocal laser scanning microscope was used to perform confocal imaging. Confocal serial sections were processed with Fiji ImageJ software (Fiji for Mac OS X; NIH, Bethesda, MD, USA) to obtain 3-dimensional projections, and image rendering was conducted with Adobe Photoshop software (Adobe Photoshop 2022, ver. 23.5.2) [26 (link)].