Z fix

Tissue collected from Siah1a^+/+::Siah2^+/+ and Siah1a^+/−::Siah2^−/− mice were fixed in Z-fix (buffered zinc formalin fixatives, Anatech) overnight. After fixation, tissues were washed twice with PBS and processed for paraffin embedding. Brain embedded in paraffin blocks were sliced at 5 µm and stained with hematoxylin and eosin. For ATF4 and CHOP staining, all cryosections were fixed with Z-fix (buffered zinc formalin fixatives, Anatech) and followed by a blacking of non-specific binding sites (Dako/Agilent Inc.) for 30 min at room temperature. Antigen retrieval was performed in a pressure cooker (Decloaking chamber, Biocare Medical) in citrate buffer (pH 6.0) and used for CHOP and ATF4 immunostaining. Antibodies/dilutions for the following markers were used in antibody diluent (Dako) overnight at 4°C. For CHOP analysis secondary antibody labeled with Alexa Fluor 488, was placed on tissue sections for 1 h at room temperature (1∶400, Molecular Probes). Nuclei were counterstained using SlowFade Gold Anti-fade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). For immunohistochemistry analysis of ATF4 the staining was visualized using an alkaline phosphatase technique (Vector red alkaline substrate kit I, Vector Laboratories).

An eyeball of an acutely-euthanized animal was fixed in an alcohol-based zinc-formalin solution (Z-fix, Anatech, Battle Creek, MI) at room temperature overnight. The eyeball was then sent to the Johns Hopkins Medical Laboratories, where it was dehydrated through a series of increasing concentrations of ethanol, embedded in paraffin, and sectioned at a thickness of 5–8 µm. Sections close to the plane of the optic disc were collected, then de-paraffinized and rehydrated by passing through Xylene and a series of ethanol solutions of decreasing concentrations. After rinsing with water, the sections were stained with haematoxylin for 3 min. Following a wash with water, the sections were cleared, rinsed and blued. The sections were then rinsed again and stained with eosin for 1 min. Finally, the slides were rinsed, dehydrated through graded alcohols, cleared by Xylene and mounted.

Mice were anesthetized, and the pancreas was rapidly dissected, fixed with Z-Fix (Anatech), and embedded in paraffin. Paraffin sections were immunostained for insulin (Abcam), glucagon (Sigma-Aldrich), and somatostatin (Abcam). Photomicrographs were obtained with a charge-coupled device camera, and the beta-cell, alpha cell, and total pancreatic areas were estimated using ImageJ software (NIH).

Femora and tibiae were de-fleshed and for each female mouse the right femur and tibiae were immediately stored in PBS and frozen without fixation for micro-computed tomography (micro-CT). The left tibiae were fixed in Z-fix (Anatech LTD) and decalcified in 10% EDTA (pH 7.4) for paraffin-embedded sectioning and histological staining. The left femur was used to harvest bone marrow-derived macrophages (BMMs) for in vitro culture.