Streptavidin horseradish peroxidase conjugate

The tissue specimens were fixed overnight in 10% neutral-buffered formalin before dehydrated in increasing concentrations of isopropyl alcohol and cleared of alcohol by xylene. The specimens were then embedded in paraffin wax in cassettes for facilitation of tissue sectioning. Standard staining with hematoxylin and eosin was performed on sections of 5 mm thickness processed from each specimen block. For immunohistochemistry, liver sections were deparaffinized and incubated in citrate buffer at 95 °C for 40 min for antigen retrieval before incubated overnight at 4 °C with the primary antibodies including anti-Zscan4 (1:200 dilution, Abclonal, A12015), anti-p-p38 (1:200 dilution, R&D, AF869), anti-p-mTOR (1:500 dilution, Cell Signaling, 2971). After 3 washes, tissue sections were incubated with biotinylated anti-mouse IgG (1:200 dilution, Vector Laboratories) for 1 h at RT then washed 3 times, after which streptavidin–horseradish peroxidase conjugates (Vector Laboratories, CA, USA) were added and the slides incubated for 45 min. After 3 washes with PBS, DAB solution (Vector Laboratories) was added and the slides were counterstained with haematoxylin.

Mouse tissue specimens were fixed overnight in 10% neutral‐buffered formalin and processed for paraffin embedding. Standard staining with hematoxylin/eosin was performed on sections of 5–8 μm thickness cut from each specimen block. For immunohistochemistry, tissue sections were deparaffinized and incubated in citrate buffer at 95°C for 40 min for antigen retrieval before incubated with primary antibodies (e.g., anticleaved Caspase 3, 1:1000) overnight at 4°C. After 3 washes with PBS, tissue sections were incubated with biotinylated secondary antibody (1:200 dilution, Vector Laboratories) for 1 h at room temperature then washed thrice, after which streptavidin‐horseradish peroxidase conjugates (Vector Laboratories) were added and the slides incubated for 45 min. DAB solution (Vector Laboratories) was then added and the slides were counterstained with hematoxylin.

Recombinant human furin (0.4 μg) was incubated with QUB-F1 (100 μM) for 45 minutes at 37°C. The sample was then denatured and reduced in Laemmli treatment buffer for 5 minutes at 95°C, resolved by SDS-PAGE, before transfer onto a nitrocellulose membrane which was blocked with a solution of Tris-buffered saline (TBS) containing 3% (w/v) bovine serum albumin and 0.1% (v/v) Tween-20. The membrane was subsequently probed with a streptavidin-Horseradish peroxidase conjugate (1:10,000) (Vector-Laboratories) in blocking solution for 1 h prior to detection by chemiluminescence (Luminata Forte Western HRP substrate (Millipore)).

Frozen kidney sections (5-μm thick) that were collected on slides were fixed and processed by hematoxylin and eosin and periodic acid schiff (PAS) staining (Sigma-Aldrich, St. Louis, MO, USA). The mesangial area was analyzed to determine the percentage of glomerular area (10 glomeruli per kidney per animal) using Image J software (National Institutes of Health, Bethesda, MD, USA). For immunohistochemistry, sections were fixed in pre-cooled acetone (−20 °C) for 5 min; after three washes in phosphate-buffered saline and a 10-min treatment with 3% H₂O₂, sections were serum-blocked and incubated with antibodies against MMP-12, CD68 (both from Abcam, Cambridge, MA, USA), FN, collagen IV (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), or nitrotyrosine (EMD Millipore, Billerica, MA, USA), followed by incubation with biotinylated anti-rabbit, anti-rat, or anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA, USA), a streptavidin-horseradish peroxidase conjugate, and finally diaminobenzidine substrate for visualization (Vector Laboratories). For glomerular assessment, the percentage of mesangial area stained as glomeruli was quantitated using 10 glomeruli per kidney per animal, using Image J software.