Entellan

In situ hybridization on paraffin sections was performed as described previously (de la Pompa et al. 1997 (link)). Briefly, E16.5 control and Dach1 eCKO embryos (n = 9 control; n = 5 Dach1 eCKO) were dissected in 1× PBS and fixed in 4% PFA overnight at 4°C. Fixed embryos were embedded in paraffin and sectioned at 10-µm thickness.
Slides were hybridized overnight with 1 μg/mL antisense Cxcl12 digoxigenin-labeled probe at 65°C. After washing in salt sodium citrate (SSC) buffer, slides were incubated with alkaline phosphatase-coupled anti-digoxigenin antibodies overnight. BM-purple (Sigma-Aldrich, 11442074001) was used to develop the color in the dark to the desired extent. Slides were then fixed in 4% PFA for 20 min, dehydrated through ethanol series and xylene (Sigma-Aldrich, 534056), and mounted using Entellan (Electron Microscopy Sciences, 14800). Images were acquired using the Zeiss Axio imager.A2 upright microscope.

TUNEL staining was performed based on the instructions of the TUNEL Kit (Roche, Germany) based on terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling (TUNEL). In brief, the slides were placed in a citrate buffer (0.1 M, pH 6.0) after deparaffinization and rehydration. We applied microwave irradiation for 1 min, double-distilled water was added to cool them down. 0.1 M Tris–HCl (pH7.5, 3% BSA, 20% normal bovine serum) immersed for 30 min in. The slides were rinsed twice with PBS, TUNEL reaction mixture was added on the sections, then incubated darkly for 60 min at 37 °C, followed by converter-POD (peroxidase) incubation at 37 °C for 30 min; DAB staining was developed and redyed with methyl green. The next step involved dehydrating the slides in 50% butanol, cleared in xylene and mounted with Entellan (Electron Microscopy Sciences). Apoptotic neurons were calculated under five randomly selected high-power fields, at least 200 cells per field. The ratio of TUNEL positive cells to total cell number was defined as apoptotic index.

Femurs of 10-month-old female mice (n = 4-7 mice per genotype) were routinely embedded in methylmetacrylate as described before (28 (link)). Sections of 6 μm were deacrylated, hydrated, and subjected to Goldner's Masson Trichrome staining. Briefly, sections were subsequently stained in hematoxylin/ferric chloride, ponceau de xylidine/acid fuchsin, orange G, and Light green solutions as described previously in detail (29 (link)). The sections were dehydrated and embedded in Entellan (Electron Microscopy Sciences, Hatfield, PA, USA). Images were taken using a NanoZoomer system and analyzed using NDP viewer (Nanozoomer 2.0 HT, Hamamatsu). Categorized bone marrow adiposity measurements based on the amount of bone marrow fat cells were performed in a blinded fashion. The following score system was used: 0 = near or complete absence of bone marrow fat cells; 1 = presence of bone marrow fat cells occupying less than half of the bone marrow cavity; 2 = presence of bone marrow fat cells occupying more than half of the bone marrow cavity; and 3 = presence of bone marrow fat cells filling nearly the entire bone marrow cavity.