3 3 diaminobenzidine dab

C2C12 cells on chamber slides were fixed with 4% paraformaldehyde for 15 min at room temperature and incubated in a blocking solution (1% BSA, 10% normal goat serum) for 1 h at room temperature. For primary antibody application, the dilution values of the anti-polyclonal antibodies were 1:500 for GABAA receptor α1 (GABAARα1) (#ab33299; Abcam, Cambridge, UK), 1:1000 for GABAA receptor γ2 (GABAARγ2) (#224003; Synaptic Systems, Goettingen, Germany), and 1:100 for both phosphorylated-Smad1/5/8 (p-Smad1/5/8) (#9511; Cell Signaling, Danvers, MA, USA) and BMP receptor 1A (#ab38560; Abcam, Cambridge, UK). The cells were incubated overnight at 4 °C. For secondary antibody application, a diluted HRP-conjugated goat anti-rabbit IgG H+L antibody (Abcam, Cambridge, UK) was used at 1:500, and the cells were incubated for 1 h at room temperature. The positive signal was detected using 3,3-diaminobenzidine (DAB; TaKaRa, Kusatsu, Japan) as a staining substrate. Sections were counterstained using hematoxylin to clearly observe tissue and cell morphology. Light micrographs were obtained using a Canon EOS Kiss X8i camera (Canon, Tokyo, Japan) on an optical microscope (OLYMPUS BX50, Olympus, Tokyo, Japan). The positive rate of cells for p-Smad1/5/8 antibody was calculated using Image J software Version 1.52a (National Institutes of Health, Bethesda, MD, USA).

An in situ apoptosis detection kit (Takara Bio, Shiga, Japan) was used for the analysis of LX-2 cells treated with HNK or vehicle according to the manufacturer’s instructions. Briefly, cells were incubated in chamber slides (SCS-N04, Matsunami Glass Ind., Osaka, Japan) with the indicated treatment for 24 h. The substrate 3,3′-diaminobenzidine (DAB) (Takara Bio, Shiga, Japan) was used for staining, which was observed with a BZ-X800 microscope. A total of 10 randomly selected fields of view at 200-fold magnification were used, and the number of the TUNEL-positive cells was averaged.

On day 1 and 3 after LI, PPU-7 cells on chamber slides were fixed with 4% paraformaldehyde for 15 min at room temperature and incubated in a blocking solution (1% BSA, 10% normal goat serum) for 1 h at room temperature. For primary antibody application, the dilution of anti-cleaved caspase-3 monoclonal antibody (Cell Signaling, Danvers, MA, USA) was used at 1:500, and the cells were incubated overnight at 4 °C. For secondary antibody application, diluted HRP-conjugated goat anti-rabbit IgG H&L antibody (abcam, Cambridge, UK) was used at 1:500, and the cells were incubated for 1 h at room temperature. The positive signal was detected using 3,3-diaminobenzidine (DAB) (TaKaRa, Kusatsu, Japan) as a staining substrate. Sections were counterstained to observe clear tissue and cell morphology using hematoxylin. Light micrographs were obtained using a Canon EOS Kiss X8i (Canon, Tokyo, Japan) camera on an optical microscope (OLYMPUS BX50, Olympus, Tokyo, Japan). The number of apoptotic cells present on a chamber slide expressed as a fraction of the total number of cells, named the “apoptotic index,” was used to evaluate apoptotic state. The number of activated caspase-3-labeled apoptotic cells and bodies was calculated in 30 high power fields (HPFs; objective X400, field diameter 640 μm). The apoptotic index was calculated as the percentage of the whole PPU-7 population.