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Demographic and clinical data were reviewed, including age, gender, height, weight, bone mineral density (BMD), and occupation history. All the data were collected by two residents from medical records or by telephone. For radiography, subjects were instructed to stand in a comfortable position, with the hips and knees fully extended [16 (link)]. Arms were flexed with the hands resting on supports at the level of their shoulders [16 (link)]. All radiographs were obtained in digital format. Parameters related to sagittal alignments were measured three times independently by a post-graduate student, a resident orthopedic surgeon, and a senior orthopedic surgeon, using Image J (Bio-Rad, Hercules, CA, USA). These investigators were blinded to patient information. Each parameter was measured by each of the three researchers, and an average value was calculated. BMD was indicated by a T score.

Tissues and immunofluorescence staining were performed according to the method previously described with a few adjustments (Li et al., 2021 (link)). Briefly, renal tissues were embedded in Tissue-Tek OCT compound, snap frozen in liquid nitrogen, and stored at −80°C till examination. To inspect the deposition of IgG, C3, C5b-9, and synaptopodin in glomeruli, 5 µm cryosections were immersed in acetone and washed in cold PBS. After blocking with 5% bovine serum albumin in PBS for 30 min, the cryosections were stained with IgG, C3, C5b-9, and synaptopodin overnight at 4°C, followed by goat anti-mouse IgG H&L (Alexa Fluor^® 488). The cryosections were observed under confocal fluorescence microscope (Zeiss LSM710, Germany). Fluorescence intensity was measured in five randomly selected fields of six cryosections by ImageJ (Bio-Rad Laboratories, Hercules, CA, United States).

Twenty to forty μg of total placental protein were loaded on Mini‐PROTEAN® TGX gel (Bio‐Rad) and resolved at 150 volts for 1 h. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio‐Rad) and blocked with 5% powdered milk in tris buffered saline solution with 0.05% tween‐20 (TBST) for 1 h at room temperature (RT). Membranes were incubated overnight at 4°C with mouse monoclonal anti‐CD68 (1:500, Abcam Cat# ab201973, RRID:AB_2936513) and rabbit polyclonal anti‐CD206 (1:800, Abcam Cat# ab64693, RRID:AB_1523910). The following day, blots were washed with TBST and incubated with 1:5000 dilutions of horse radish‐peroxidase conjugated secondary antibodies (Goat Anti‐Mouse IgG Bio‐Rad Cat# 1706516, RRID:AB_2921252; Goat Anti‐Rabbit IgG Bio‐Rad Cat# 1706515, RRID:AB_2617112) for 1 h at RT. The blots were developed using Clarity ECL Western Substrate (Bio‐Rad) and imaged using ChemiDoc™ XRS+ Imaging System (Bio‐Rad). Membranes were permanently stained with 1% Amido Black for total protein lane quantification. Band expression and total protein expression were analyzed by densitometry (ImageJ, Bio‐Rad). Placental protein expression for CD68 and CD206 was standardized to total participant pooled protein lysate samples.

The Bcl-2 and Bax protein level in left ventricle sample was determined by western-blotting. Antibodies for Bcl-2 (sc-492) and Bax (sc-493) were purchased from Santa Cruz Biotechnology, INC., CA, USA. The protein bands were captured with an image processor using the Bio-Rad software (CA, USA) and the intensity of the bands was measured using ImageJ (NIH, USA).