Image pro plus 6

After deparaffinization and processing for antigen retrieval, the blank colon sections were incubated overnight at room temperature with anti-rabbit ZO-1 antibody (1:500, catalog no. GB11195; Servicebio, China) and Occludin antibody (1:200, catalog no. GB11149; Servicebio) separately. Sections were then incubated with anti-rabbit IgG secondary antibody (KPL, MA, USA) for 50 min at room temperature. After washing with PBS, the DAB Envision kit (catalog no. K5007; Dako, Copenhagen, Denmark) was used to develop color; ZO-1 and Occludin appear brown, while nuclei are blue. Images of each colon section were obtained in tripartite by experienced staff who were blind to the experiment under ×200 magnification using a Leica DMRBE microscope and were analyzed using Image Pro Plus 6.0, according to the method previously described [47 (link)]. The integrated optical density (IOD) values were log10 transformed.

Fixed hearts were laterally cut along the central plane to create a cross section on the left and right sides of the right ventricle. Fixed small intestines were sagittally sectioned. Heart and small intestinal tissues were embedded in paraffin and sliced (5 mm thick). Myocardial morphology was detected by haematoxylin and eosin (H&E) staining. Heart samples were stained with Masson's trichrome to analyse myocardial fibrosis. Images were then captured using a Leica Microsystems microscope and quantified using Image-Pro Plus 6.0.

The concentration of intracellular Ca²⁺ was determined using a cell-permeable fluorescent calcium indicator, Fluo 4-AM. Cells were treated with DMEM-F12 and 10% bovine serum for 24 h and washed 3 times with D-Hanks balanced salt solution without Ca²⁺ (Ca²⁺-free HBSS). Subsequently, cells were loaded with 2μmol/l Fluo 4-AM for 30 minutes at 37 °C in the dark, then washed 3 times with Ca²⁺-free HBSS to remove the extracellular Fluo 4-AM. The solution was replaced with HBSS with Ca²⁺ before testing. Imaging was performed using the Leica SP8 Confocal Inverted Microscopy (Leica, Mannheim, Germany) and analyzed with Image-Pro Plus 6.0. We used the measured average fluorescence intensity of each cell in the field (F), normalized to the non-specific background fluorescence (F0) to obtain the fluorescence intensity (F/F0)^{22 (link)}.

Cells cultured on 20-mm-diameter Glass Bottom Cell Culture Dish (Cell-Nest) were fixed with 4% paraformaldehyde for 20 min, washed with PBS three times, and permeabilized with 0.25% Triton-X 100 for 10 min. The permeabilized cells were blocked with PBS containing 3% BSA for 1 h at 37 °C and stained with 1:200 dilution of anti-NUP358 antibody, anti-p24 antibody and blocking buffer for overnight at 4 °C. Cells were washed three times with PBS, then were incubated with a 1:500 dilution of a Dylight 549-conjugated goat anti-mouse IgG antibody (Invitrogen), Dylight 488-conjugated goat anti-rabbit IgG antibody and blocking buffer for 1 h at 37 °C and then washed with PBS three times. Cells were stained with Hoescht 33342 (Invitrogen) diluted to a concentration of 1 mg/mL for 3–5 min, following three washes with PBS. Subsequently, samples were mounted for fluorescent microscopy by using the Antifade Mounting Medium (beyotime). Images were obtained with Leica confocal microscope (Leica-LCS-SP8-STED, Germany) using a 63× objective and were analyzed with Leica Application Suite X software and Image Pro-Plus 6.0.

After deparaffinization of paraffin sections, rehydration was done, and the endogenous peroxidase was blocked by 3% H₂O₂. Then,the sections were incubated with normal goat serum at 37°C for 20 min and subsequently incubated with rabbit polyclonal antibodies against TLR4 (1:500), NF-κB (1:200), IL-6 (1:400) and TNF-α (1:500) at 4°C overnight. After being rinsed, the sections were incubated with a horse radish peroxidase (HRP)-labeled secondary goat anti-rabbit antibody for 30 min. Antigens were visualized with a diaminobenzidine (DAB) approach. Immunostained paraffin sections were photographed under a light microscope (Leica, Germany) and a proportion of positive immunostained sections were analyzed using Image-Pro Plus 6.0.

Lungs were excised from the thoracic cavity, inflated, fixed with 4% neutral buffered polyformaldehyde and then cut into slices. Tissues were embedded in paraffin and stained with hematoxylin&eosin (H&E), Periodic acid-Schiff (PAS), or Masson’s trichrome (MT). Pictures were taken using a DM 4000B Microscope (Leica), and analyzed with Image-Pro Plus 6.0 to determine the average optical density (AOI) of the PAS and MT stained sections.