Z3781

Lysates and microsomal fractions were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose
membranes according to standard procedures. Membranes were probed with
commercial antibodies against β-Gal (Promega z3781), HO-1 (AbCam,
ab13243), or in-house polyclonal antisera against rat P450s, CYP1A1, CYP2B1,
CYP3A1, and CYP4A1. These have previously been shown to cross-react
specifically with the murine counterparts of their target P450s. The antibody
for Nqo1 was obtained from Abcam (ab2346); the antibody for Gsta1/2 was a kind
gift from Professor John Hayes (Kelly
et al., 2000; O’Connor et al., 1999 (link)).
Commercial antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(Sigma G9545), Lactate dehydrogenase (LDH) (Abcam, ab2101), and calrecticulin
(AbCam, ab2907) were used as loading controls. Immunoreactive bands were
visualized on x-ray film developed using an Xograph Compact X4 automatic film
processor (Xograph Imaging Systems, Gloucestershire, UK).

Samples for immunohistochemical analysis and hematoxylin and eosin (H&E)
staining were fixed in a solution of 1% paraformaldehyde and Gurr®.
After overnight fixation at room temperature, they were stored in 70%
ethanol. When required, they were processed using a Shandon Citadel 2000 tissue
processor (Thermo Scientific) and embedded in paraffin wax in a Shandon
HistoCentre 3 embedding center (Thermo Scientific). Sections
(5 µM) were cut using a Shandon Finesse 325 microtome (Thermo
Scientific). The DakoCytomation EnVision® Dual Link System-HRP (DAB+)
kit (Dako Ltd, High Wycombe, UK) was used to carry out immunohistochemical
analysis of 5-µM sections according to manufacturer’s instructions.
Sections were stained using antibodies against β-Gal (Promega z3781) or
HO-1 (AbCam, ab13243) at a dilution of 1:100, and counterstained with
hematoxylin.
For H&E staining, 5-µM liver sections were deparaffinized in xylene,
rehydrated in decreasing alcohol concentrations, stained with H&E,
dehydrated in increasing alcohol concentrations, and mounted using DPX mounting
media (Sigma), all according to standard procedures. The sections were
photographed under bright field conditions on a Zeiss Axiocam microscope; the
resulting images were processed with AxioVision software (Zeiss).