Image pro plus 6

A cerebral block containing the hippocampus and prefrontal cortex was fixed in 10% neutral-buffered formalin overnight and then embedded in paraffin. Coronal 10-μm sections were prepared and subjected to immunohistochemistry staining. First, paraffin sections were dewaxed and placed in EDTA buffer (pH 8.0) to repair antigens. Second, sections were washed in 0.01% Triton X-100 in phosphate-buffered saline (PBS-T) and blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature. Then, they were incubated overnight at 4 °C in the appropriate primary antibody, anti-Iba1 (1:100; WAKO). Next, the sections were incubated with the appropriate secondary antibody, anti-rabbit IgG (1:400; Jackson) for 2 h at room temperature. Glial reactivity is characterized by an increase in the number of cells and an alteration in cell morphology (rounding of the cell bodies and thickening of processes), which leads to an increase in Iba1 (ionized calcium-binding adaptor molecule 1) labelling with increasing glial reactivity. An increase in the integrated intensity/pixel area for Iba1 staining was interpreted to signify microglial reactivity. The number of positively stained microglial cells per view was counted using microscopy at ×200 magnification. Images were captured using the Olympus BX5 imaging system and quantified using Image-Pro Plus 6.0 software.

A549 and H1299 cells (3 × 10⁵ cells/well) were seeded in 6-well plates for 24 h and a clear cell-free line was manually created by scratching the confluent monolayers with a 100 μL pipette tip in each well. The wounded monolayers were washed three times with PBS and photographed using an inverted microscope (Olympus, Hamburg, Germany) as the picture for 0 h. Then, the cells were co-treated with TGF-β1 (5 ng/mL) for 48 h in combination with ViceninII (2.5, 5 and 10 μM) for the last 24 h. TCell migration was captured as images at 48 h. The migration area was counted by Image-Pro Plus 6.0 software (Bethesda, MD, USA).

Hearts were immediately excised, weighed, washed in cold phosphate-buffered saline, and cut into several pieces. The left ventricular sections were fixed in 4% paraformaldehyde at room temperature for 24 h. Then, the samples were dehydrated and embedded in paraffin blocks, and cross-sections at 5 μm thick were obtained. For evaluation of heart pathological morphology and myocardial fibrosis, hematoxylin and eosin (H&E) staining and Masson's trichrome staining were conducted following the manufacturer's instructions (Servicebio, Wuhan, China). For evaluation of cardiomyocyte size, paraffin-embedded sections were deparaffinized, rehydrated in xylene and graded ethanol, and then subjected to wheat germ agglutinin (WGA) staining according to the manufacturer's protocol (Servicebio, Wuhan, China). All images were acquired using an optical microscope (Olympus, Tokyo, Japan) and analyzed by Image-Pro Plus 6.0 software.

The cultured cells were prepared and stained according to the manufacturer's instructions, and the apoptotic cells were measured by the Apoptosis Fluorescein Detection Kit (Millipore, USA). Briefly, the cells were washed 3 times with PBS and fixed in 4% paraformaldehyde for 5 minutes. Cells were treated with proteinase K (20 μg/ml) for 20 minutes and incubated with terminal deoxyribonucleotidyl transferase and deoxyuridine triphosphate for 60 minutes. Nuclei were stained with DAPI (Invitrogen, USA). The results were observed using a fluorescence microscope (OLYMPUS, Japan) and calculated using Image-Pro Plus 6.0 software (Maryland, USA).

Total protein from mice skin tissues or melanocytes was extracted using total protein extraction reagent (Beyotime, Shanghai, China) according to the manufacturer’s instructions, and the concentration was measured using a nucleic acid/protein analyzer. Two hundred micrograms of protein lysate from each sample were size-separated by SDS-PAGE electrophoresis and transferred onto nitrocellulose filter membranes (Boster, Wuhan, China). The membranes were blocked in 5% skimmed milk powder (Boster, Wuhan, China) at room temperature for 1 h and then were probed with the primary antibody diluted in Tris-buffered saline-Tween (TBST) overnight at 4 °C. The next day, the membranes were washed 3 times in TBST for 10 min each and incubated with HRP-conjugated second antibody (1:10,000 (v/v), Boster, Wuhan, China) at 37 °C with horizontal shaking for 1 h. Subsequently, the membranes were washed 6 times in TBST for 5 min each, and the proteins were visualized via a super ECL chemiluminescence solution (Boster, Wuhan, China). The Western blot results were analyzed using Image-ProPlus 6.0 software (Olympus, Hatayaga, Japan) to measure the area and gray value for each target band. The target protein expression level was normalized relative to the corresponding internal reference level in each lane.

First, the heart sections were washed with PBS, and the primary antibody was diluted in FACS buffer and added into a hydrated chamber (anti-CD19 rabbit pAb [Servicebio]) overnight at 4 °C. Afterwards, sections were washed with PBS and stained with secondary antibody diluted in PBS for 1 h at 4 °C (Cy3 conjugated goat anti-rabbit IgG [Servicebio]). Sections were subsequently washed with PBS and incubated with DAPI solution for 10 min at room temperature. The sections were washed again with PBS, and then spontaneous fluorescence quenching reagent was added and the sections were incubated for 5 min. Finally, the sections were washed in running tap water for 10 min. Immunofluorescence images were obtained under a microscope (Olympus) at 400 times magnification and analysed with Image-Pro Plus 6.0 software.