K4063

Twenty-five micrograms of protein was separated by the electrophoresis of polyacrylamide denaturing gels and transferred onto polyvinylidene fluoride membrane. The primary antibodies, rabbit–antimouse caspase-3 (1:400, 9662, CST, Danvers, USA), rabbit–antimouse high-mobility group box-1 protein (HMGB1, 1:1,000, 3935, CST), mouse–antimouse proliferating cell nuclear antigen (PCNA, 1:1,000, M0879, DAKO, Glostrup, Denmark), rabbit–antimouse erythropoietin receptor (EPOR, 1:1,000, PAB18350, Abnova, Taiwan), rabbit–antimouse properdin (1:1,000, AB186834, Abcam, Cambridge, USA) and mouse–antimouse β-actin (1:5,000, A5441, Sigma, Dorset, UK), were applied to the membranes overnight at 4°C, followed by the incubation of horseradish peroxidase-labeled secondary antibody (goat–antirabbit/mouse, K4063, DAKO) and developed by enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, USA). The ratio of target protein to β-actin in volume density, as an endogenous loading control, was calculated for each detection, and then, the fold change of detected protein in the experimental group against the WT sham control was obtained as final results (27 (link)).

Placental samples (3 µm thick) from 10% formalin-fixed, paraffin-embedded material were deparaffinized in xylene and dehydrated through ethanol solutions. Endogenous peroxidase activity was then blocked by methanol containing 0.3% hydrogen peroxidase for 30 min. The sections were incubated with anti-hCG (1:500, Dako, A0231) overnight at 4 °C and then incubated with Envision + Dual Link, Single Reagents, HRP Rabbit/Mouse (undiluted solution, Dako, K4063) for 30 min at room temperature. The reaction products were visualized by 3,3-diaminobenzidine tetrahydrochloride, and the sections were counterstained with hematoxylin.

Mice were exposed to RF or sham treatment. Skin was dissected at indicated times, fixed in formalin, and then subjected to paraffin sectioning. Paraffin sections were subjected to standard hematoxylin and eosin staining to visualize microscopic structures or Trichrome staining to visualize dermal collagen levels. For IHC, paraffin sections were deparaffinized and then subjected to heat-induced epitope retrieval with antigen unmasking solution (H-3300, Vector Laboratories). Endogenous peroxidase was depleted with 0.3% hydrogen peroxide. Tissue sections were then incubated with SuperBlock (Thermo Fisher Scientific) and then rabbit anti-mouse polyclonal HSP70 antibodies (1:100, AF1663, R&D Systems) at 4 ˚C overnight. Tissue sections were then rinsed in TBS for three times followed by incubation with HRP-conjugated anti-rabbit secondary antibodies (K4063, DAKO). After washing in TBS, DAB substrate was added and reaction was stopped 2 min later. Sections were counterstained with 20% Gill III hematoxylin, dehydrated, and then cover slipped. Images were taken under Nikon Eclipse E600 microscope by an investigator unknown of sample groups.