To pro

Paraffin embedded submandibular gland tissue sections were processed for immunofluorescence analysis as previously described [22 (link)]. Primary antibodies used at the indicated dilutions include ΔNp63 (1:50, Cell Signaling Technology, D2K8X), K5 (1:100, gift from Dr. Julie Segre), K14 (1:100 [45 ]), alpha-smooth muscle actin (Sma) (1:200, Sigma, 1A4), Aqp5 (1:100, Alomone Labs), K7 (1:50, Abcam), Mist1 (1:100, Abcam), Troma-III (K19, 1:50, Development Studies Hybridoma Bank), Ki67 (1:100, Leica Biosystems, MM1), and Cleaved Caspase-3 (1:100, Cell Signaling Technology, D175). Sections were stained with TOPRO (Invitrogen) and mounted using VECTASHIELD Antifade Mounting Medium (Vector Laboratories) and imaged using an Andor Dragonfly Spinning Disk Confocal Microscope with Fiji [46 (link)]. Microscopy data in this study was acquired at the Optical Imaging and Analysis Facility, School of Dental Medicine, State University of New York at Buffalo.

For IHC assessment, a tissue micro-array was constructed using cores of 0.6 mm in diameter. Three zones were used for each tumor: intratumoral, invasion front, and peritumoral. Three cores were used for each zone. Peritumoral zones were not obtained for two tumors. Sequential 3-μm sections were cut and colored by HES or used for immunohistochemistry.
IHC was also performed on tissue sections from formalin-fixed paraffin-embedded tissue blocks from WT and OSM-KO mice. Serial sections of 3 μm were cut. Immunohistochemistry was performed using a BenchMark automated staining system (Ventana Medical System, Tucson, AZ) for CD3 (Dako, A0452, 1:200), CD68 (Ventana, 790-2931, ready to use), and MPO (Dako, IS511, ready to use). Images were taken using a camera fixed to the microscope (Leica Application suite, version 4.4). Quantification of inflammatory cells was performed using assisted counting software (Visilog Noesis).
For immunofluorescence studies, tumor sections were fixed in acetone/methanol (20/80) and stained with anti-OSM (R&D system, MAB4951, 1:50), followed by staining with a goat anti-rat-IgGAF555 secondary antibody (Thermofisher, A-21434, 1:100). Cell nuclei were stained with TOPRO (Invitrogen). Image acquisition was performed with an Olympus FV1000 confocal microscope using FlowView software (ImageUP platform, University of Poitiers).

Precursor or mature astrocyte cultures were fixed at 2, 14, 19, 21 and 28 DIV with 4% paraformaldehyde (Sigma-Aldrich, Cat. #P6148), and immunostained with antibodies against CD44, GFAP, S100β, ALDH1L1, Cx43, EAAT2, AQP4, Islet-1, MAP2, Nestin and Iba1, as described in Table 1. Antibody binding was visualized with the appropriate fluorescent secondary antibodies as described in Table 2. Immunolabeled APCs or astrocytes were documented on an inverted Nikon Eclipse Ti-U microscope equipped with a SPOT Pursuit™ USB Camera CCD (14-bit), Epi-FL illuminator, mercury lamp, and Sutter Smart-Shutter with a lambda SC controller. Cells were photographed using a ×40 objective; positive cells were counted offline within five randomly chosen fields, and the percentage of positive cells compared to the total number of TO-PRO (Invitrogen, Cat. #T3605) positive cells was calculated.

Evaluation of the expression and localization of the TJ protein claudin‐5 after Aβ peptide treatment in CMEC cells was evaluated by immunocytochemistry (ICC) as previously described (Fossati et al., 2010). Briefly, cells were plated on collagen‐coated glass chamber slides (Thermo Fisher Scientific) and were grown in EBM‐2 0.25% FBS +bFGF 1:2000 to facilitate the development of tight junctions. After cells were confluent for 5 days, cells were treated for 24 h with 10 μM of Aβ42 oligomers, Aβ42 fibrils, or freshly solubilized Aβ42, washed with PBS, and subsequently fixed for with 4% paraformaldehyde at room temperature. Claudin‐5 was visualized by incubation with anti‐claudin‐5 mouse monoclonal antibody (Invitrogen), followed by an incubation with Alexa Fluor 488‐conjugated anti‐mouse IgG (Invitrogen) and nuclei visualized by counterstaining with To‐pro (Invitrogen). Images were then acquired using Nikon Eclipse Ti inverted fluorescence microscope with deconvolution. Specificity of the antibody detection was assessed by omission of the primary antibody during the ICC procedure.