Hrp labeled secondary antibody

Immunohistochemical staining of HMGB1 in renal biopsies was performed as described previously [21 (link)]. In brief, kidney sections (4 μm) were used for all staining experiments. Sections were deparaffinised. Next, endogenous peroxidase blocking and antigen retrieval was performed. Slides were incubated with rabbit anti-HMGB1 antibody (Abcam, Cambridge, UK). Subsequently, slides were incubated with HRP-labeled secondary antibodies (DakoCytomation, Glostrup, Denmark). Next, slides were counterstained with hematoxylin. The renal staining of HMGB1 was evaluated by the Image Pro Plus analysis software 6.0 (Media Cybernetics, Silver Spring, MD). Cellular distribution of HMGB1 was determined in the kidney by counting one hundred nuclei in three brightfield pictures and scoring both HMGB1-positive (brown) and HMGB1-negative (blue) nuclei. Results are expressed as the percentage of negative cells. In each assay, a primary isotype Ig control has been used as the negative control.

Cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 0.5% DOC, 0.1% SDS, 1% NP-40, and 150 mM NaCl) with protease and phosphatase inhibitors (#4693159001 and #4906837001; Roche), followed by sonication at the highest setting with a pulse of ± 30 s. After centrifugation at 16,000g for 30 min, lysates were collected in a fresh tube and protein concentration was determined using BCA assay (Pierce). 6× loading buffer was added to lysates, and they were boiled at 95°C for 10 min. Equal amounts of lysates were loaded on 4–20% precast gradient gels (Bio-Rad) and separated at 80–100 V. Proteins were blotted using PVDF membrane (Bio-Rad) and blocked for 1 h at RT. Membranes were incubated overnight with primary antibody followed by washing. For detection, HRP-labeled secondary antibodies (DAKO) followed by incubation with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) were used, or LI-COR Biosciences secondary antibodies (IRDye 680 or IRDye 800) were used followed by detection by Odyssey Imaging Systems or Bio-Rad Laboratories ChemiDoc Imaging Systems.

Tumor slides were stained according to standard IHC protocols. Briefly, slides were blocked with 10% bovine serum albumin (Sangon) for 1 h, incubated with PCNA antibodies (Abcam, ab18197) overnight at 4 °C, and then incubated with HRP-labeled secondary antibodies (Dako) for 1 h at 25 °C. Antibodies were detected using diaminobenzidine substrate chromogen (DAB). All slides were counterstained with hematoxylin before dehydration and mounting.

A quarter of a kidney was homogenized using a IKA^® tissue homogenizer in 500 μL RIPA buffer (Sigma Aldrich, St. Louis, MO, USA) containing Complete^® protease inhibitors (Roche, Basel, Switerland). Subsequently, a Pierce BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA) was performed to assess the absolute quantity of protein in each sample. Next 10–40 μg of total protein lysate was loaded per lane on precast Any-kD acrylamide gels (Bio-Rad, Hercules, CA, USA) for gel electrophoresis. Using the Trans-Blot^® Turbo™ Transfer system (Bio-Rad), the protein was transferred to Nitrocellulose (Bio-Rad) membranes for further analysis. Membranes were blocked and antibodies incubated o/n at 4 °C in 5% skim milk powder (Nutricia, Zoetermeer, Netherlands) in PBS. The appropriate HRP-labeled secondary antibodies (Dako, Glostrup, Denmark) were incubated for 1 h at room temperature, followed by extensive washing with PBS. Band-visualization was achieved using SuperSignal West-Dura^® Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA, USA) for HRP. Next, exposure on UltraCruz^® Autoradiography Film or KODAK-XAR film and development by a Konica developer followed. Finally, band intensities were quantified using ImageJ 1.x [62 (link)].

Protein samples were extracted from murine heart ventricular tissues with 10 mM Tris-Maleate buffer (pH 7.0). Protein concentration was measured by BCA Protein Assay Reagent Kit (Thermo Fisher Scientific). Twenty μg of protein samples were loaded on a 4–15% SDS-PAGE gel (Bio-Rad) for separation and transferred to PVDF membranes with Trans-Blot Turbo system (Bio-Rad). After blocking with Blocking One (Nacalai tesque), the membranes were incubated overnight at 4 °C with the custom-made anti-RBM20 affinity-purified antibody described above (1 µg/ml), anti-Tnni1 (1:500, rabbit polyclonal, Proteintech), anti-Tnni2 (1:500, rabbit polyclonal, Proteintech), or anti-GAPDH (1:10,000, mouse monoclonal, Santa Cruz Biotechnology). HRP-labeled secondary antibodies (1:10,000, Dako) were used for detecting the specific bands. Chemiluminescense signals (GE Healthcare) were detected by using iBright CL1500 (Thermo Fisher Scientific), and analyzed using iBright Analysis Software (Thermo Fisher Scientific).

Western blot protein lysates were prepared in 1× RIPA buffer (150 mm sodium chloride, 1.0% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mm Tris, pH 7.5) containing PhosSTOP™ phosphatase inhibitors and cOmplete™ EDTA‐free protease inhibitors (Roche). DNA in the protein samples was sheared by sonication and the amount of protein was estimated using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Lysates were separated on 4–20% Mini‐PROTEAN® TGX™ Gels (Bio‐Rad, Hercules, CA, USA) and transferred by wet blotting to PVDF membranes (Merck Millipore, Burlington, MA, USA), or via semi‐dry transfer to nitrocellulose membranes (Bio‐Rad). Unspecific antibody binding was blocked with 5% non‐fat dry milk in TBST. Incubation for the primary antibodies was performed overnight at 4 °C in either 5% non‐fat dry milk, or in 5% BSA for the phospho‐specific antibodies. For detection, HRP‐labeled secondary antibodies (DAKO/Agilent, Santa Clara, CA, USA) followed by incubation with Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) were used. Alternatively, LI‐COR Biosciences secondary antibodies (IRDye 680 or IRDye 800) were used, followed by detection by Odyssey® Imaging Systems or Bio‐Rad Laboratories ChemiDoc Imaging Systems.