Mas coated slides

Serial sections (2 μm) were mounted on MAS-coated slides (Matsunami Glass Ind.,Ltd, Osaka, Japan) and processed for heat-induced antigen retrieval by autoclave with citrate buffer (pH 6.0) for OLIG2 or EDTA buffer (pH 9.0) for glial fibrillary acidic protein, CD34, and α-SMA, followed by treatment with 3% H₂O₂ in PBS for 10 minutes and were washed thrice in PBS. After blocking in 10% normal goat serum in PBS for 1 hour, the sections were incubated in moist chambers overnight at 4°C with antibodies to glial fibrillary acidic protein (rabbit polyclonal, prediluted; Dako Cytomation, Glostrup, Denmark), OLIG2 (rabbit polyclonal, dilution 1:100; IBL, Fujioka, Japan), CD34 (mouse monoclonal, dilution 1:50; Dako Cytomation), and α-SMA (mouse monoclonal, dilution 1:100; Dako Cytomation). After washing with PBS, the corresponding secondary antibody reaction was conducted. 3,3′-diaminobenzidine (DAB) solution was used for visualization. The slides were counterstained with hematoxylin, were dehydrated, and were cover-slipped.

ISH was performed as previously described^{61 (link)} with some modifications. Briefly, fixed uterine tissues were paraffin-embedded and paraffin sections (6 µm) were mounted on MAS-coated slides (Matsunami Glass Industries, Osaka, Japan) under RNase-free conditions. Sense or antisense digoxigenin (DIG)-labeled RNA probes for Gp130 (Il6st) and suppressor of cytokine signaling 3 (Socs3) were purchased from Genostaff. The sections were deparaffinized, rehydrated, and post-fixed in 10% neutral buffered formalin (NBF) for 30 min at 37 °C, followed by the treatment with 0.2% hydrogen chloride and 5 µg/mL proteinase K (FUJIFILM Wako Pure Chemical, Osaka, Japan) for 10 min at 37 °C, respectively. Hybridization was performed with DIG-labeled probes (250 ng/mL) in a humidified chamber at 60 °C overnight. The slides were washed after hybridization, then treated with blocking reagent (Genostaff) for 15 min and alkaline phosphatase-conjugated anti-DIG antibody (1:2000; Roche Diagnostics, Basel, Switzerland) for 1 h at room temperature. The signals were detected by 4-nitro-blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Roche Diagnostics) in a humidified container for 12 h at 4 °C. The sections were counterstained with Kernechtrot solution (Muto Pure Chemicals, Tokyo, Japan). Signals detected by sense probe was used as a control for background levels.

Mice were deeply anesthetized with sodium pentobarbital and then perfused via the aortic cone with phosphate-buffered saline (PBS), followed by 4% PFA containing 0.2% saturated picric acid in PB. The lumbar region of the spinal cord was removed and postfixed in the same fixative for 2 h at 4 °C, after which it was dehydrated and embedded in paraffin. Sections were cut at 7 μm, mounted on MAS-coated slides (Matsunami Glass), and hybridized with digoxigenin (DIG)-labeled probes, as described previously64 (link). To prepare the DIG-labeled OPN probe, a portion (189–985) of the mouse OPN sequence (NM_009263.3) was subcloned into pBluescript SK vector (Stratagene), and antisense and sense probes were transcribed using T7 and T3 RNA polymerase, respectively. The sense probe gave no hybridization signal. After color development using NBT/BCIP, sections were briefly washed in PBS and then processed for immunohistochemical detection of Iba1 using DAB as the chromogen, as described in Experimental Procedures.

This study was approved by the Human Investigation Committee (No. 1202) of our institution (Table 1).
Formalin‐fixed and paraffin‐embedded (FFPE) samples of OLP tissues were used for hematoxylin and eosin staining and IHC. Biopsied tissue specimens were obtained from patients with oral mucosal lesions showing reticular, erosive, or atrophic changes who were examined at the Division of Oral and Maxillofacial Surgery (Department of Oral and Maxillofacial Reconstructive Surgery, School of Dentistry, Iwate Medical University).
Ten FFPE samples containing normal‐appearing oral mucosa, which were obtained by excising cysts, benign tumors, or other diseases, were used as controls. The mean age of the 10 patients (four males and six females) was 59 years (range: 31‐79 years).
Specimens were cut into 4‐μm‐thick serial sections and mounted onto MAS‐coated slides (Matsunami, Osaka, Japan). The slides were deparaffinized, rehydrated, and used for hematoxylin and eosin staining and IHC.