Sc 11418

ZytoChem-Plus AP Kit (Fast Red) (Zytomed Systems, Berlin, Germany) was used for immunohistochemical localization of research target-proteins in formalin-fixed, paraffin-embedded lung tissue sections from patients with sporadic IPF (n = 5) and organ donors (n = 5), according to the manufacturer´s instructions and previous published work [31 (link)]. In the following, the primary antibodies used for IHC are listed, including the sources and dilutions: rabbit polyclonal for human alpha-smooth muscle actin [α-SMA] (1:200, Abcam, ab5694), rabbit monoclonal for human cytokeratin-5 [KRT5] (1:200, Abcam, ab75869), rabbit polyclonal for human survivin (1:200, Abcam, ab24479), mouse monoclonal for human phospho-STAT3 [Y705] (1:25, Cell Signaling Technology, #4113S) and rabbit polyclonal for human HDAC4 (1:50, Santa Cruz, sc-11418). As control experiments, the first antibody was omitted on some sections during staining procedures. Immunostained lung sections were scanned with a scanning device (Nano-Zoomer, Hamamatsu), and examined histopathologically using the ´NDP.view2 software´ at 50×, 100×, 200× and 400× original magnification.

Protein expression in the hippocampus was separated using 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples from each group contained an equivalent amount of nuclear or total protein per well. The electrophoretic proteins were transferred onto a polyvinylidene difluoride membrane (Immobilon-P membrane; Millipore; Bedford, MA, USA) and probed with specific antibodies against Ki67 (1:1000, Ab16667, Abcam), SOX2 (1:1000, Ab97959, Abcam), Nestin (1:1000, Ab6142, Abcam), PAX6 (1:1000, MAB5552, Merck Millipore, Middlesex, MA, USA), Doublecortin (DCX, 1:1000, Ab18723, Abcam), and HDAC4 (1:1000, sc-11418, Santa Cruz Biotechnology Inc.). Membranes were then incubated with the appropriate horseradish peroxidase–conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). The specific antibody–antigen complex was detected using an enhanced chemiluminescence Western Blot detection system (Thermo Fisher Bioscience). The amounts of detected protein were quantified using ImageJ software (NIH, Bethesda, MD, USA). The purity of the nuclear and total fractions was verified by assessing the expression of TBP and β-actin (Millipore), respectively.

Protein (20 µg) was run on NuPAGE Novex 10% Bis-Tris Gels (Invitrogen-Life Technologies) and transferred onto 0.45-μm PVDF membranes (Millipore, Stonehouse, United Kingdom). The membranes were blocked in 5% milk and incubated overnight at 4°C with HDAC4 (1:500, sc-11418; Santa Cruz Biotechnology, Dallas, TX, USA), HDAC5 (1:1000, H4538; Sigma-Aldrich,), HDAC9 (1:500, ab18970; Abcam, Cambridge, United Kingdom), α-tubulin (Sigma-Aldrich) or H3 (1:10,000, ab1791; Abcam). After they were washed, the membranes were incubated in secondary antibody for 1 h (1:5000, horseradish peroxidase–conjugated anti-rabbit or anti-mouse; GE Healthcare, Little Chalfont, United Kingdom). The signal was detected using an enhanced chemiluminescence prime kit (GE Healthcare) and visualized with a UVP GelDoc-It Imaging system (Ultra-Violet Products, Cambridge, United Kingdom).