2 methylbutane

Twenty-four hours after the behavioral assessment, animals were euthanized with an intraperitoneal injection of pentobarbital (55 mg/kg, Exagon®, Med’Vet, France) for neuroinflammatory evaluation through ionized calcium-binding adapter molecule 1 (IBA-1) immunofluorescence staining. The procedures were similar to those we previously described^{50 ,51 (link)}. After, mice were transcardially perfused with 0.9% NaCl, followed by 4% paraformaldehyde fixative (PFA, Roth®, Karlsruhe, Germany) dissolved in 0.1 M phosphate buffer (PB, pH 7.4). Extracted brains were postfixed overnight at 4 °C in the same fixative and then cryoprotected for 24 h at 4 °C by immersion in a 15% sucrose solution (D(+)- Saccharose, Roth®, Karlsruhe, Germany) in a 0.1 M PB. Finally, the brains were rapidly frozen via immersion in isopentane (2-methylbutane, Roth®, Karlsruhe, Germany) and sliced into serial coronal 30-µm-thick sections.

Brains were collected and fixed in a 4% paraformaldehyde fixative solution in 0.1 M phosphate buffer (pH 7.3-7.4) cryoprotected in 30% sucrose and then quickly frozen in 2-methylbutane (Roth, Karlsruhe, Germany) cooled with dry ice to −40°C. Coronal serial sections (30 µm) were collected and stored at −80°C. Nissl stain was performed with thionin and photographed at 1.253× with a Sony DFW-X700 digital camera (Sony, Tokyo, Japan) coupled to an Olympus BX60 light microscope (Olympus, Hamburg, Germany).

All animal protocols complied with the guidelines for animal care approved by the German Animal Care Committee (Landesamt für Naturschutz, Umwelt und Verbraucherschutz Nordrhein-Westfalen (LANUV) located in Recklinghausen, Germany; https://www.lanuv.nrw.de). Permit numbers for respective animal protocols are: 84-02.04.2012.A092 (approved 15 August 2012) for CCl₄ injection experiments and 84-02.04.2015.A028 (approved 4 March 2015) for hepatocyte isolation from murine livers.
In our study, we used 6–8-week-old C57BL/6 wild type and Lcn2^−/− mice to investigate CCl₄-induced ER stress. To do so, we injected mice intraperitoneally with 0.8 mL CCl₄/kg body weight diluted in mineral oil twice a week for eight consecutive weeks. Thereafter, the animals were euthanized. Blood was drawn by heart puncture and liver specimens snap-frozen in liquid nitrogen for later analysis. Frozen tissue sections were preserved in Tissue-Tek (Sakura Finetek Europe B. V., Alphen aan den Rijn, The Netherlands) solved in in ice-cold 2-methylbutane (Roth, Karlsruhe, Germany). The samples were stored at −80 °C, or alternatively fixed in 4% buffered paraformaldehyde for subsequent immunohistological stainings.

Mice were anesthetized by intraperitoneal injection of 16% sodium pentobarbital solution (Narcoren, Merial, Hallbergmoos, Germany, 5 µl/g body weight) and were transcardially perfused with 4% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.3. The spinal cord was removed two hours after fixing, post-fixed overnight at 4 °C and then immersed in 15% sucrose solution in 0.1 M cacodylate buffer, pH 7.3, for 1 day at 4 °C. Afterwards the tissue was frozen for 2 min in 2-methyl-butane (isopentane, Carl Roth, Karlsruhe, Germany) pre-cooled to −80 °C. For sectioning, the spinal cord segment was attached to a cryostat specimen holder using TissueTek (Sakura Finetek Europe, Zoeterwoude, The Netherlands). Serial transverse or parasagittal sections of 25 μm thickness were cut on a cryostat (Leica CM3050, Leica Instruments, Nußloch, Germany) and picked up on Super Frost Plus glass slides (Roth, Karlsruhe, Germany). Sampling of sections was always done in a standard sequence so that four sections 250 μm apart were present on each slide. Immunohistochemistry was performed as described earlier61 (link).