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To induced NSCs and GDNF/NSCs differentiation, neurospheres were adhered to coverslips precoated with poly-L-lysine (10 μg/mL, Sigma) in six-well plates in DMEM/F12 medium in the absence of bFGF, EGF, and N2 supplement but with 10% fetal bovine serum (FBS) for 7 days. The cultures were fixed with 4% paraformaldehyde for 30 min and incubated with neuronal specific markers microtubule-associated protein 2 (MAP2, 1 : 100, rabbit, Abcam, UK), astrocytes specific markers glial fibrillary acidic protein (GFAP, 1 : 100, rabbit, Abcam), and oligodendrocyte specific marker galactocerebroside (GalC, 1 : 50, rabbit, Chemicon, USA) overnight at 4°C, followed by appropriate biotinylated secondary antibodies (1 : 100, Wuhan Boster Biological Technology, China) and horseradish peroxidase- (HRP-) streptavidin (Boster) for 30 min at 37°C. Immunoreactivity was visualized with diaminobenzidine (DAB, Boster). Counterstaining by hematoxylin was also performed. A negative control was performed using the same procedures without primary antibody. The ratio of MAP2⁺, GalC⁺, or GFAP⁺ cells to total cells was quantified in 5 regions in 3 coverslips per group.

Anti-A20, anti-elastin antibody was obtained from Santa Cruz Technology (Santa Cruz Biotech, Santa Cruz, CA, USA). Primary polyclonal antibodies against phospho-IKKβ (Ser180/181), phospho-IκBα (Ser32/36), IKKβ, IκBα, and NF-κB p65 are from Cell Signaling Technology. Anti-CD45, anti-CD68 and anti-CD20 were from Boster, China. Anti-β-actin, anti-MMP-2, anti-MMP-9 were purchased from Bioss, China. Calcium chloride anhydrous (CaCl₂) and zinc sulfate heptahydrate (ZnSO₄·7H₂O) were purchased from Sigma. Diaminobenzidine (DAB) and strept-avidin biotin complex (SABC) immunohistochemical kit were purchased from Boster (Wuhan, China). Fetal bovine serum and Dulbecco's modified Eagle's medium (DMEM) were purchased from Hyclone (Logan, Utah, USA).

The rat testes tissue samples were fixed in 10% formalin, embedded in paraffin, and cut into 6 μm thin sections. The tissue sections were deparaffinized and then rehydrated with decreasing concentrations of ethyl alcohol (100%, 100%, 95%, 95%, 85% and 75%). Then, endogenous peroxidaseactivity was blocked by incubating the samples in 3% hydrogen peroxide followed byantigen retrieval. The specimens were then blocked with 5% bovine serum albumin (BSA) in room temperature for 30 min, followed by incubation with rabbit anti-CD34 (1:100 dilution; catalog no. BA3414; Boster, Wuhan, China), mouse anti-αSMA (1:100 dilution; catalog no. BM0002; Boster, Wuhan, China) antibodies at 4°C for 24 hours. Negative contral group was performed by 0.01 PBS (pH=7.4) at the same condition. After washing, the sections were incubated with biotinylated anti-rabbit/anti-mouse IgG (Boster Bio-Technology, Wuhan, China) for one hour at room temperature. The sections were then incubated with avidin-biotinylated peroxidase complex. Peroxidase activity was determined by staining with diaminobenzidine (DAB, Boster Bio-Technology, Wuhan, China). The nuclei were stained with hematoxylin.

Human PDAC tissues were fixed in 4% paraformaldehyde (pH 7.0–7.6, containing 0.1% DEPC) and then embedded in paraffin. A digoxigenin-labeled miR-361-3p oligonucleotide probe (5′-AAATCAGAATCACACCTGGGGGA-3′) and an ISH kit (MK10503) were obtained from Boster Biological Technology (Wuhan, China). Slides were deparaffinized and incubated for 20 min at room temperature with Pepsin; subsequently, the sections were prehybridized in a humid chamber at 38–42 °C for 2 h. Then, the tissues were hybridized overnight with a miR-361-3p probe at 38-42 °C. After hybridization, the slides were washed with graded-diluted sodium citrate buffer (SSC) at 37 °C for 40 min, followed by incubation with an antibody against digoxigenin at 37 °C for 60 min. These sections were then incubated with SABC-POD kit (Boster, Wuhan, China), and hybridization signals were visualized using diaminobenzidine (DAB) (Boster, Wuhan, China). Finally, the tissues were counterstained with hematoxylin, mounted and imaged.

Visual analysis of plaque morphology was performed as follows. Briefly, near-confluent (85 to 90%) DEF monolayer cells in 6-well plates with glass coverslips were infected with BAC-CHv and the gE mutant and revertant viruses at an MOI of 0.01. The viruses were allowed to penetrate for 2 h at 37 °C in 5% CO₂. Thereafter, the culture medium was removed, and the cells were washed three times with PBS (pH 7.4). The DEF cells were incubated for 24 h in MEM containing 1% methylcellulose and 2% NBS. The culture medium was removed again, and the cells were washed three times with PBS. The cells were then fixed with 4% paraformaldehyde overnight at 4 °C, washed three times with PBS-T and permeabilized with 30% H₂O₂ and methanol at a ratio of 1 to 50 for 30 min. The cells were incubated with 5% BSA (Biotechnology) for 30 min and then incubated with the rabbit anti-CHv polyclonal antibody and goat anti-rabbit IgG antibody conjugated to SABC. After washing three times with PBS-T, the cells were colorized by the addition of diaminobenzidine (DAB, BosterBio). Photographs of viral plaques were taken at 40× magnification, and 100 randomly selected plaques were imaged for the viruses under consideration.