Af3318

The diseased tissue was cut into pieces, lysate added, centrifuged, and the protein concentration detected using a BCA protein assay kit (Beyotine, Wuhan, China). An electrophoretic gel was prepared and the protein samples were mixed with 5 × loading buffer and treated at 100 °C for 5 min. An amount of 100 μg of cell protein was collected from each well, and electrophoresis was performed with a constant current power source of 15 mA per gel. The transfer tank was filled with electro-transfer solution to start the film transfer and the color-developing reagent system was added to the protein membrane^{20 (link)}. The ratio of the target to GAPDH light density was regarded as the relative concentration of protein expression. The primary antibodies were SYK (AF3314; Affinity Biosciences), p38 (AF4001; Affinity Biosciences), JNK (AF3318; Affinity Biosciences), PI3K (AF3241; Affinity Biosciences), and ERK (AF1015; Affinity Biosciences).

One rat testis per rat was removed and fixed in Bouin’s solution for 24 h. The testes were then dehydrated in graded ethanol, embedded in paraffin, and sectioned (4 µm in thicknesses). The sections were stained with hematoxylin-eosin (HE) dye and observed under a light microscope (Leica DM500, 100x).
Next, the sectioned paraffin-embedded slides of rats’ spermary per group were deparaffinized and rehydrated with xylene and gradient alcohol. The slides were then incubated with primary antibodies [PI3K p85 (60225-1-Ig, Proteintech), p-PI3K p85 (AF3242, Affinity), AKT (60203-2-Ig, Proteintech), p-AKT (AF0832, Affinity), JNK (66210-1-Ig, Proteintech), p-JNK (AF3318, Affinity), p53 (60283-2-Ig, Proteintech), and p-p53 (AF3075, Affinity)] overnight at 4°C following an antigen repair and closure. Next, the slides were incubated with a secondary antibody labeled with horseradish peroxidase (HRP) at room temperature for 1 h. The slides were then treated with 3,3-diaminobenzidine (DAB) and hematoxylin, dehydrated with alcohol, and sealed with neutral gum. Finally, slides’ images were captured by the Olympus BX53 fluorescence microscope (40x).

Total protein was extracted with RIPA lysis buffer. Protein concentration was determined by using the Bradford method. Equal amounts of protein were electrophoresed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk and then incubated with primary antibodies, including anti-CTRP3 (1:1500, orb182796, Biorbty Ltd), anti-LAMP1 (1:1000, DF7033, Affinity), anti-JIP2 (1:750, DF3260, Affinity), anti-p-JNK (1:1000, AF3318, Affinity), anti-JNK (1:1000, AF6318, Affinity) and anti-GAPDH (1:20000, 10494-AP, Proteintech) at 4°C overnight. The membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:10000, 19003-1-AP, Proteintech). GAPDH was used as an internal control. The signals were detected by using an ECL kit (Pierce Biotech, Rockford, IL, USA). Subsequently, the protein bands were analyzed with Image J 1.43 software.

The mice were administered 1% pentobarbital sodium and decapitated. Then, bilateral hippocampal tissues were immediately collected. SH-SY5Y cells were collected after exposure. Cells and lysed tissues in RIPA buffer supplemented with protease and phosphatase inhibitors were collected. Protein concentrations were measured with BCA reagent (P0006, Beyotime Biotechnology, China). Protein samples (20 μg) were separated by SDS‒PAGE (10% separation gel) and transferred to a PVDF membrane (Merck and Co., Inc., Whitehouse Station, NJ, United States, Germany). After blocking with TBST containing 5% nonfat milk for 1 h, the membranes were incubated with Gpx4 (ab125066, 1:1000; Abcam), Hmox-1 (ab189491, 1:2000; Abcam), P2rx7 (ab259942, 1:1000, Abcam), phospho-Erk1/2 (AF1015, 1:1000; Affinity Biosciences), Erk1/2 (AF0155, 1:1000; Affinity Biosciences), phospho-p38 (AF4001, 1:1000; Affinity Biosciences), p38 (AF6456, 1:1000; Affinity Biosciences), phospho-Jnk (AF3318, 1:1000; Affinity Biosciences), and Jnk (AF6318, 1:1000; Affinity Biosciences) overnight at 4 °C. The next day, following three washes in TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (A0208, 1:2000, Beyotime) or anti-mouse IgG (A0216, 1:2000, Beyotime) at room temperature for 1 h. The protein levels were normalized to GAPDH (#5174, 1:1000, Cell Signaling Technology).

Western blot assay was performed as described by Sale et al [19 (link)]. Total proteins were extracted from cells using RIPA lysate (R0010, Solarbio) containing phenylmethylsulfonyl fluoride protease inhibitor (PMSF, P0100, Solarbio). Then, protein concentration was detected with a BCA kit (PC0020, Solarbio). After separated by 5-10% SDS-PAGE, transferred to the PVDF membrane, blocked by nonfat milk (A600669, Sangon Biotech, China), proteins on the membrane were incubated overnight at 4°C with the primary antibodies: iNOS (1: 1000, A0312, Abclonal, China), COX-2 (1: 2000, A1253, Abclonal), MMP-1 (1: 2000, A1191, Abclonal and 1: 1000, DF6325, Affinity), MMP-3 (1: 2000, A11418, Abclonal), MMP-13 (1: 2000, A11148, Abclonal and 1: 1000, AF5355, Affinity), p-ERK1/2 (1: 1000, AF1015, Affinity, China), ERK1/2 (1: 1000, AF0155, Affinity), p-p38 (1: 500, AF4001, Affinity), p38 (1: 500, AF6456, Affinity), p-JNK (1: 1000, AF3318, Affinity), JNK (1: 500, AF6318, Affinity), p-p65 (1: 1000, AF2006, Affinity), p65 (1: 1000, AF5006, Affinity), and GAPDH (1: 10,000, 60,004-1-Ig, Proteintech, China). Next, the membrane was incubated with appropriate secondary antibody. Finally, membranes were detected by Western electrogenerated chemiluminescence (ECL) Substrate (D1010, Solarbio) and the images were analyzed by Gel-pro analyzer software (Media Cybernetics, CA, USA).

The diseased tissue sections were dewaxed, incubated at room temperature for 5–10 min, washed with distilled water, and soaked in PBS for 5 min. Drops of primary antibody were added, and the tissue sections incubated overnight at 4 °C. The tissue was then washed three times with PBS, biotin-labeled secondary antibody was added after 5 min, and the tissue sections were incubated at 37 °C for 30 min^{19 (link)}. The tissue was again washed three times with PBS, DAB stained, rinsed with water, hematoxylin stained, mounted, and imaged under a microscope (Olympus CX41, Tokyo, Japan). Polyclonal Rabbit IgG (ab37415, Abcam) was selected for isotype control antibody, which immunohistochemically showed all negative in 4 LGBLEL patients. The primary antibodies used for the immunohistochemical staining of proteins related to FcεRI signaling pathway were spleen tyrosine kinase (SYK) (AF3314; Affinity Biosciences, Beijing, China), p38 (mitogen-activated protein kinase, MAPK) (AF4001; Affinity Biosciences), c-Jun N-terminal kinase (JNK) (AF3318; Affinity Biosciences), phosphoinositide 3-kinase (PI3K) (AF3241; Affinity Biosciences) and extracellular signal-regulated kinases (ERK) (AF1015; Affinity Biosciences).