Il 17

Brain samples were homogenized and sonicated in lysis buffer. Total protein was measured by a NanoDrop apparatus (MaestroGen, Las Vegas, NV, USA). 20 μg protein were size-fractionated using any-kD Mini-Protean TGX gel electrophoresis and then transferred to a nitrocellulose membrane, and membranes were blocked and washed in TBS-T, and incubated overnight with AC, ACC1, ACC2, PC, PCC, MCC, presynaptic synapsin I, postsynaptic PSD95, PSD93, IL-17, IL-6, TNF-α, NFkB, GFAP, GAP43, ICAM-1, BDNF, CXCL 16, OPG and MMP-9 antibodies (Abcam, Cambridge, UK). The following day, membranes were washed and then incubated with horseradish peroxidase-conjugated goat anti-rabbit (Abcam, Cambridge, UK) or goat anti-mouse (Abcam, Cambridge, UK) secondary antibody (diluted 1:1000) for 1 h at room temperature. Protein loading was controlled with an anti-β-actin antibody (Abcam, Cambridge, UK). Protein levels were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MD, USA), corrected with values determined on β-actin blots, and expressed as relative values compared with the control group.

Prostatic sections were fixed overnight in a 10% buffered paraformaldehyde solution. The tissues were ethanol-dehydrated and embedded in paraffin wax. Sections of 5 µm were cut with microtome, deparaffinized at 70°C for 20 min, incubated 5 minutes each in xylene, ethanol-xylene, and 100% ethanol. Finally, the samples were washed with distilled water to be processed for immunohistochemical analysis. Immunolabeling was done by using Mouse/Rabbit PolyDetector Plus HRP/DAB (BSB 0259; BioSB) Kit according to the manufacturer's instructions. The antibodies used were TNF-α (50 µg/mL) (Abcam 1793; Massachusetts, USA), IFN-γ (50 µg/mL) (Abcam 7740; Massachusetts, USA), CD45 (1 µg/mL) (Abcam 10558, Massachusetts, USA), IL-17 (1 µg/mL) (Abcam 79056, Massachusetts, USA), IL-6 (74 µg/mL) (Abcam 6672; Massachusetts, USA), IL-4 (0.5 µg/Ml) (Abcam 11524; Massachusetts, USA), and PCNA (100 µg/mL) (Santa Cruz-53407, Texas, USA). After the immunohistochemistry procedure, microphotographs were taken and the relative expression percentages were semiquantified in the Image-Pro Premier software 9.1.

Gradient concentrations of xylene and alcohol were used for paraffin section dewaxing and hydration, and then the sections were heated for antigen retrieval. After cooling to room temperature, the sections were soaked in 3% H₂O₂ solution to inactivate the endogenous peroxidase (only for immunohistochemistry). All sections were treated with 10% goat serum for 30 min to block the antigen and then incubated with primary antibodies at 4°C overnight. The secondary antibody (Zhongshan Biology Co. Ltd, China) corresponding to the primary antibody was incubated at room temperature for 30 min. For immunohistochemistry staining, diaminobenzidine was used for antibody staining and hematoxylin (Beyotime, China) for nuclear staining. For immunofluorescence staining, DAPI was used for nuclear staining. The results were observed and photographed under a microscope (Olympus, Japan).
The primary antibodies used are described as follows: IL-1β (1:200, Abcam, United Kingdom); IL-6 (1:200, Abcam, United Kingdom); IL-8 (1:200, Bioworld, United States); K1 (1:200, Abcam, United Kingdom); K6 (1:200, Thermo Fisher Scientific, United States); TNF-α (1:200, Abcam, United Kingdom); IL-23 (1:200, Abcam, United Kingdom); CCL20 (1:100, Abcam, United Kingdom); IL-22; IL-17 (1:250, Abcam, United Kingdom); IFN-γ (1:200, Abcam, United Kingdom); and BrdU (Abcam, United Kingdom).

Western blotting with proteins of the cortex and myocardium was performed as described previously (Mlyniec et al. 2014a , b (link)). To confirm equal loading of the samples on the gel, the membranes were re-probed with an antibody specific to GAPDH as an internal control. The specific primary antibodies used included rabbit polyclonal antibodies against CAIII (1:1000; abcam), Beclin-1 (1:200; Santa Cruz), LC3 (1:1000; abway), P53 (1:1000; abcam), IL-17 (1:1000; abcam), NF-κB (1:200; Santa Cruz), GAPDH (1:2000; Boster). Finally, the X-ray films were developed and fixed in a dark room.

Immunohistochemistry was performed as described previously (Tu et al. 2017 (link)). Tissues samples of the cortex and myocardium were fixed with 4% polyformaldehyde, embedded in paraffin, cut into slices (thickness, 10 μm), dewaxed and hydrated. Then the slices were incubated with 3% hydrogen peroxide, washed with PBS, blocked with 10% normal goat serum at 37 °C for 30 min, incubated with rabbit polyclonal antibody for CAIII (1:200; Abcam), P53 (1:100; Abcam), IL-17 (1:1000; Abcam), in PBS containing 3% BSA overnight at 4 °C, followed by incubation with biotinylated secondary anti-rabbit antibody at 37°Cfor 45 min. Immunohistochemical staining followed by diaminobenzidine staining was subsequently performed. Finally, all sections were dehydrated sequentially with 75% ethanol for 5 min, 85% ethanol for 5 min, ethyl alcohol twice for every 5 min, and xylene for 6 min. They were then covered on slides for image analysis.

Sections 5 μm thick of the lacrimal gland were deparaffinized, hydrated, processed in antigen retrieval solution (Abcam Inc., Cambridge, UK), and exposed to 3% H₂O₂ solution (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) for 30 min. For immunohistochemical analysis, the slides were incubated with primary antibodies against cluster of differentiation 4 (CD4; Novus Biologicals, Littleton, CO, USA), interleukin-17 (IL-17; Abcam Inc., Cambridge, UK), and tumor necrosis factor alpha (TNF-α; Abcam Inc., Cambridge, UK) 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., Yongin, Korea). The stained slides were photographed using an imaging system (Thermo Fisher Scientific). The quantitative analysis of histological staining for CD4, IL-17, and TNF-α was performed using the “threshold tool” of ImageJ^® (National Institutes of Health, Bethesda, MD, USA).