H2o2 solution

The method used by Watanabe et al. [44 (link)] was used with the following modifications. The reaction mixture was composed of 0.02 mL of EDTA solution (56.5 mM), 0.01 mL of ^the sample, 0.02 mL of 3% H₂O₂ solution (Sigma H1009), and 0.02 mL of catalase solution (4000-fold diluted, Sigma C3515) (all reagents except the tested sample were diluted in TRIS buffer, pH 7.0, 1 mol/dm³). The volume was completed to 0.31 mL by the same buffer solution. In a blank sample, DMSO (Sigma D4540) replaced the tested sample. The background of the sample was measured in a mixture composed of 0.01 mL of the sample completed to 0.31 mL by the buffer. The absorbance was read at 240 nm directly after the mixing and after 5 min of incubation (room temperature). The decreased absorbance (depletion of H₂O₂) in the tested and blank samples was compared. The calibration curve was produced using eleven H₂O₂ solutions (0.5693–5.693 mM/dm³). The results are expressed in % inhibition and as H₂O₂ depletion (mmol of depleted H₂O₂/dm³ min).

3% H₂O₂ solution and dimethyl sulfoxide were obtained from Sigma (St. Louis, MO). RPMI-1640 culture medium, 0.05%Trypsin-EDTA, and fetal bovine serum (FBS) were from Gibco (Grand Island, NY, USA). One step TUNEL apoptosis assay kit, puromycin dihydrochloride, bicinchoninic acid assay kit, glutathione peroxidase kit, catalase assay kit, DAPI staining solution, DAF-FM diacetate kit, dihydroethidium, superoxide dismutase, and malondialdehyde assay kit were obtained from Beyotime (Shanghai, China). Anti-Bax, anti-Bcl-2, and anti-Caspase-3 were from Abcam (Cambridge, MA, USA). Anti-PI3K, anti-Akt, antiphosphorylation-PI3K, and antiphosphorylation-Akt were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Immobilon-PSQ transfer membrane was obtained from Millipore (Billerica, MA, USA). An Annexin V/FITC apoptosis detection kit was from BD Biosciences (San Diego, CA, USA). PI3-kinase LY 294002 was purchased from MedChemExpress LLC (New Jersey, USA). Lipofectamine™ 3000 transfection reagent was purchased from Thermo Fisher Scientific, Inc. (Invitrogen, USA). Lentivirus control and PI3K shRNA (U6-MCS-Ubiquitin Cherry-IRES-puromycin) were purchased from GeneChem (Shanghai, China).

For immunohistochemical analysis, 5-μm thick sections of the lacrimal gland and eyeball were deparaffinized, hydrated, processed in antigen retrieval solution (Abcam, Inc., Cambridge, UK) and exposed to 3% H₂O₂ solution (Sigma-Aldrich Chemical Co.) for 30 min. The slides were incubated with primary antibodies against cluster of differentiation 3 (CD3, Abcam, Inc.), CD4 (Novus Biologicals, Littleton, CO, USA), F4/80 (Abcam, Inc.), interleukin-6 (IL-6, Abcam, Inc.), interleukin-17 (IL-17; Abcam, Inc.) and tumor necrosis factor alpha (TNF-α; Abcam, Inc.) for 1 h. Subsequently, the sections were incubated with secondary antibodies (DAKO Corp, Glostrup, Denmark) for 40 min, followed by probing with diaminobenzidine chromogen, and counterstained with Mayer’s hematoxylin (YD Diagnostics Co.). The stained slides were photographed using an imaging system (Thermo Fisher Scientific). The quantitative analysis of histological staining performed using “threshold tool” of ImageJ^® (National Institutes of Health, Bethesda, MD, USA).

Immunohistochemistry for collagens type I 5-μm histological sections were deparaffinized, rehydrated and subjected to enzymatic digestion with 0.4% pepsin (Sigma, USA) diluted in 0.5 N acetic acid for 30 min at 37 °C. For proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA), the 5-μm histological sections were deparaffinized, rehydrated and subjected to antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 5 min in a pressure cooker. After blockading endogenous peroxidase with 6% H₂O₂ solution (Merck) for 30 min, the slides were incubated in a humidified chamber overnight at 4 °C with the following rabbit primary antibodies: collagen type I (#600-401-103, Rockland, USA), PCNA (clone PC10, cod. M0879, DAKO, USA) and α-SMA (clone 1A4, cod. A2547 Sigma, USA). The slides were then incubated with the complex Super Picture Polymer Detection kit (Life Technologies, USA) for 30 min at 37 °C. The reaction was visualized with 3’3 diaminobenzidine chromogen and counterstained with Harris hematoxylin. The negative controls were performed by omitting the primary antibodies. Counterstaining was performed using Carazzi’s hematoxylin. Slides (n = 175) slides were examined under light microcopy, in a Zeiss microscope coupled with a 176 Image Evaluation System (Kontron 300).