Image pro plus 6.0 system

Animals were sacrificed after three or six weeks of treatment. The tibia and femur were dissected and fixed in 4% paraformaldehyde for 24 h, decalcified in 10% ethylenediamine tetraacetic acid (EDTA) and embedded in paraffin. Tissue sections (4 μm) were mounted on common slides for staining with hematoxylin and eosin, toluidine blue and Masson’s trichrome as described previously (16 (link),17 (link)). Cartilage histopathological features were analyzed using the scoring system modified by Mankin et al (18 (link)) (score range 0–12, from normal to complete disorganization and hypocellularity). Synovium histopathology was evaluated according to Yoshimi’s histological grading (score range 0–18, between normal and most severe reaction) (19 (link)). The Image-Pro Plus 6.0 System (IPP) image analysis system (Media Cybernetics, Rockville, MD, USA) was used for quantitative analysis. The positive index was calculated as the integral of the optical density. All sections were randomized and evaluated by a trained observer who was blinded to the treatment groups.

The DRG fixed in 4% buffered formaldehyde was embedded in paraffin. The tissue block was sectioned at a thickness of 4-μm, and mounted on poly lysine coated slides. Four serial slides were deparaffinized and rehydrated sequentially, followed by the Streptavidin–Biotin Complex immunohistochemistry procedure as follows. The serial slides were incubated with rabbit-anti-CGRP (Abcam Inc., Cambridge, MA-02139-1517, USA; 1:100 diluted in PBS), and PBS (the negative control), respectively, and, sequentially, for 24 h at 4°C. Then, they were treated with secondary antibodies (SA1022-anti-rabbit IgG Kit, Wuhan Boster Biological Technology Ltd., Wuhan, China). Avidin-biotin complex Staining (Wuhan Boster Biological Technology Ltd., Wuhan, China) was visualized with diaminobenzidine (DAB) for 1 min at room temperature. All sections were dehydrated in graded ethanol series, made transparent in xylene, coverslipped, and air-dried. The slides were visualized with the images of the stained areas under a light microscope (Nikon ECLIPSE 80I, Nikon Corporation, and Tokyo, Japan), and 3 fields of each slide were observed with a 20 × objective lens. The numbers of CGRP positive cells were counted with the Image-Pro plus 6.0 system (Media Cybernetics, Inc., Bethesda, MD, USA). The mean values calculated from three sections represented the CGRP positive cells per rat.

Samples were taken from the same part of the livers of BA mice, fixed in 10% formalin, embedded in paraffin, and routinely sectioned, followed by hematoxylin and eosin (HE) staining, as well as Masson staining to observe the histological changes and collagen fiber distribution in the liver, respectively. The area, perimeter, mean optical density, integral optical density, and mean grayness of liver collagen fibers were quantified by the Image-Pro Plus 6.0 system (Media Cybernetics, Rockville, MD, United States) according to the criteria for staging pathological liver fibrosis in China (18 (link)). The degree of liver fibrosis was assessed by measuring the hydroxyproline content of the samples by the acid hydrolysis method (19 (link)).

The paraffin sections (4-μm-thick) were soaked in 3% H₂O₂ for 10 min after dewaxing with xylene and alcohol baths. The sections were then soaked in antigen retrieval solution for 5 min. Then, PBS with 10% sheep serum (Gibco, Grand Island, NY, USA) was added to the section to block the non-specific sites. The primary antibody against ZMIZ2 (Abclonal) was added and then incubated at 4 °C overnight, followed by the incubation with the corresponding secondary antibody (Bioss). The images (200 × magnification) were photographed and the positive area density was analyzed using an Image-Pro Plus 6.0 System (Media Cybernetics Corporation, USA).