Sc 575

For histopathological examination, the liver tissue sections were stained with haematoxylin-eosin (H&E). Sirius red was used to stain for collagen. Immunohistochemistry was performed using 4–5 mm paraffin-embedded thick liver sections, and observed under a photomicroscope. Immunofluorescence staining were observed under a laser scanning confocal fluorescence microscope (Carl Zeiss, Jena, Germany). Antibodies were used as follow: FOXA2 (ab108422, Abcam), α-SMA (BM0002, Boster), α-SMA (MO85129, Dako), CHOP (sc-575, Santa Cruz) and Cleaved Caspase 3 (Asp175,9664, Cell Signaling). Areas of positive stained sites were measured by using image analyses software Image-Pro Plus 6.0 (Media Cybernetics).

HK-2 cells were transfected with eGFP or TDAG51 bound to GFP (TD-GFP). 48 h after transfection, cells were fixed in 4% paraformaldehyde, and immunofluorescently stained with CHOP antibodies (red; sc-7351; Santa Cruz) and a fluorescently labeled secondary antibody. Cell nuclei were stained with DAPI (blue). HK-2 cells were also transfected with pcDNA3.1 control vector or pcDNA3.1 vector with human CHOP gene. 72 h after transfection, cells were fixed and immunofluorescently stained with TUNEL (red; Roche) for apoptosis and CHOP antibodies (green; sc-575; Santa Cruz) with a fluorescently labeled secondary antibody. Cells were imaged using an Olympus IX81 Nipkow scanning disc confocal microscope.

Sections (4 µm thick) were cut, air-dried, and deparaffinized through a series of xylene and graded ethanol (100%–70%). Heat-Induced Epitope Retrieval was performed in Retrieve-All-2 buffer (Signet Laboratories, Dedham, MA) pH 10, for 30 min followed by 0.1% Triton-X for 10 min at room temperature for CHOP staining [12] (link). No antigen retrieval was used for GRP78 staining [32] (link). Tissues were stained for CHOP to determine if treatment with TM or 4-PBA resulted in modifications in the expression of this transcription factor. Sections were incubated with either a rabbit anti-CHOP antibody (1∶40, sc-575; Santa Cruz Biotechnology) or a goat anti-GRP78 antibody (1∶40, sc-1050; Santa Cruz Biotechnology). The primary antibody was detected using a species-specific secondary antibody conjugated to an Alexa dye at 647 nm excitation wavelength, producing an emission maximum at 668 nm in the far-red region of the spectrum (1∶500; Invitrogen). This emission wavelength was used due to the high levels of autofluorescence in the renal tubular epithelium at the typical emission wavelengths for FITC (520 nm) or tetramethylrhodamine (580 nm). Sections were incubated with 100 ng/ml DAPI to label cell nuclei and mounted with Permafluor (Thermo Scientific).