Formaldehyde

Prepared cells were used for immunostaining with anti-cytokeratin antibody (Nichirei, Tokyo, Japan) and anti-vimentin antibody (DAKO, Carpinteria, CA, USA). Cultured cells were washed with phosphate-buffered saline (PBS) and then treated with PBS containing 10% formaldehyde (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 20 min. After washing with PBS containing 1% TritonX-100 (Nacalai tesque, Kyoto, Japan) for 5 min, the cells were treated with a blocking reagent (PBS containing 1% BSA; Sigma-Aldrich, USA) for 30 min at 37°C. The blocked cells were washed three times with PBS containing 0.05% Tween20 (FUJIFILM Wako Pure Chemical Co., Japan) for 10 min. The washed cells were probed with a primary antibody and incubated 4°C overnight. Subsequently, after washing with PBS containing 0.05% Tween20 for 10 min three times, the cells were incubated with secondary antibody (Goat anti-mouse immunoglobulin G [IgG]/fluorescein isothiocyanate [FITC]) (Nordic Immunology, Tilburg, Netherlands) for 30 min at 37°C in the dark. The stained cells were observed under a fluorescence microscopy (IX70; OLYMPUS, Tokyo, Japan).

The excised kidney tissue was fixed with 3.7% formaldehyde (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) diluted in PBS and then embedded in paraffin to prepare 3 μm tissue sections. Tissue sections were subjected to Periodic Acid-Schiff (PAS) staining according to the method of Schaart et al. [41 (link)] and observed under an optical microscope (BX53F, OLYMPUS, Tokyo, Japan) to assess the proliferation of glomerular mesangial cells and mesangial matrix in glomeruli. Then, mesangial proliferation scores were calculated based on the percentage of glomeruli with mesangial cells’ proliferation and increased mesangial matrix among 50 glomeruli in PAS-stained sections [42 ,43 (link)]. Additionally, immune-fluorescence staining of IgA in the kidney tissues was detected by using standard immunohistochemical techniques with IgA-specific antibodies. The primary antibody was Purified Rat Anti-Mouse IgA (BD Biosciences, San nose, CA, USA). Secondary antibodies were Goat Anti-Rat IgG (Alexa Fluor-488) (Invitrogen, Carlsbad, CA, USA). The blocking procedure was performed by using Normal Donkey Serum (Jackson ImmunoResearch, West Grove, PA, USA). Fluorescence immunostaining was performed by using these antibodies. Stained sections were observed under an all-in-one fluorescence microscope (BZ-X700; Keyence, Osaka, Japan).

Cells were fixed with 80% acetone at −20°C for 10 min or 2.5% formaldehyde (Wako; 063-04815) at room temperature for 10 min. formaldehyde-fixed cells were permeabilized with 0.5% Triton X-100 in PBS. Fixed cells were blocked in 2% BSA in PBS for 30 min. Cells were incubated at 37°C for 1 h with each of the primary and secondary antibodies. For chromosome spreads, HT1080 tetR–EYFP–CENP-B N21 cell line (Fig. 8C) was treated with 350 nM TN-16 (Wako) (Kitagawa et al., 1995 (link)) for 3 h in the culture medium. Mitotic cells were harvested, incubated in 0.075 M KCl for 10 min on ice, and then spread on a cover-glass using a Cytospin3 centrifuge (Shandon). The subsequent immunostaining and fluorescence in situ hybridization (FISH) were performed to confirm HAC formation according to a previously reported method (Ikeno et al., 1998 (link); Ohzeki et al., 2002 (link)). For Halo-tag labeling, cells were cultured in the presence of 10 nM Halo-TMR-Ligand (Promega; G8251) or 10 nM Halo-Biotin-Ligand (Promega; G8281).

Immunohistochemical staining analysis was performed as previously reported [5 (link), 14 (link)]. Briefly, the samples were fixed with 3.7% formaldehyde (FUJIFILM Wako Pure Chemical Industries) and permeabilized with PBS containing 0.2% Tween-20 (FUJIFILM Wako Pure Chemical Industries). Samples were then blocked with PBS containing 4% Block Ace (DS Pharma Biomedical) and 0.2% Tween-20 and then incubated with primary antibodies (mouse anti-NGFR (1:200; Advanced Targeting System) and rabbit anti-SOX10 (1:500; Abcam)) overnight at 4°C. Samples were then washed with PBS containing 0.2% Tween-20 thrice and then incubated with secondary antibodies (anti-mouse Alexa Fluor-488 and anti-rabbit Alexa Fluor-555; 1:1000; Thermo Fisher Scientific). For nuclear staining, 0.2 μg/ml Hoechst 33342 (Dojindo Molecular Technologies) was added to the samples. The samples were placed on the stage of the inverted microscope (IX81; Olympus), and fluorescence was observed with an electron multiplying charge-coupled device camera (iXon; Andor). The observed images were analyzed using ImageJ software (National Institutes of Health; available at http://imagej.nih.gov/ij/).

Three different concentrations of formalin (2%, 4%, and 8%) were prepared by diluting a saturated aqueous solution of formaldehyde (37%) (Wako, Japan) with sterile physiological saline solution.

Urea was purchased from Kanto Chemicals (Tokyo, Japan). Phosphoric acid, sodium iodide, indium chloride, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol, ethanol, chloroform, formaldehyde, and Triton X-100 were purchased from Fujifilm Wako Pure Chemical Co (Osaka, Japan). Phosphate buffered saline (PBS) was purchased from Thermo Fisher Scientific K.K. (MA, USA). N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG; Sunbright DSPE-020CN) was purchased from NOF America Corporation (NY, USA). All the reagents were used without further purification, and handled safely without touching directly, in particular for Phosphoric acid of high concentration.