Decalcifying solution lite

The PIP joints were collected from each group at 8 weeks after the first immunization. The tissues were fixed in 10% neutral buffered formalin solution, decalcified using Decalcifying Solution-Lite (Sigma, St. Louis, MO, USA), and embedded in paraffin. Sections of 4- to 5-μm thickness were cut, dewaxed using xylene, dehydrated in an alcohol gradient, and stained with hematoxylin and eosin (H&E) and safranin O.

All chemicals used in the study were of analytical grade and were either procured from an Indian manufacturer (SRL India, HiMedia chemicals) or obtained from Sigma Aldrich (St Louis, MO), Thermo Fisher Scientific Inc (USA) etc. Mannan oligosaccharide (MOS), 1,3,5-trihydroxybenzene, phloroglucinol, D-xylose, ethylene diaminetetracetic acid (EDTA), BCA kit, hematoxylin, eosin, thiobarbituric acid, BSA, Mops, Sucrose, TBA, DTNB, NEM, neutral buffered formalin, decalcifying solution-Lite, were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO, USA. Phosphate buffer saline (PBS), acetic acid, hydrochloric acid, and all other chemicals obtained were of analytical grade from SRL India. Reagents for hematology analyzer (Isotonac 3, Hemolynac 5, Hemolyzing Reagent, Hemolynac 3, Cleanac 3) were procured from Nihon Kohden, Japan.

Formalin fixed femurs were treated with Decalcifying solution-Lite (Sigma) according to the manufacturer’s instructions. Formalin-fixed spleen, thymus and decalcified femurs were embedded in paraffin. Microscopic slides with a 4 μm thickness were prepared from the paraffin-embedded tissues and then subjected to hematoxylin-eosin (H&E) staining. Histological examination and capture of digital images were performed under a light microscope (BX41, Olympus).

One leg was randomly selected from each mouse and dissected for histology. The knee joints were collected and fixed in 4% paraformaldehyde solution. Fixed joints were decalcified in Decalcifying Solution Lite (Sigma‐Aldrich, St. Louis, MO, USA) for 6 h and embedded in paraffin. Joint tissues were sectioned by a microtome, and sections (5 μm) had been hydrated in 100%, 90%, 70%, and 50% ethanol in distilled water (DW) for 5 min each. Hydrated samples were stained with haematoxylin and eosin (H&E) (Sigma‐Aldrich, St. Louis, MO, USA) for morphological analysis and measurement of the infiltration of immune cells, or safranin O and fast green (Sigma‐Aldrich, St. Louis, MO, USA) for determining cartilage damage. Stained samples were dehydrated in 50%, 70%, 90% and 100% ethanol and xylene and mounted by using balsam. They were observed by using DM750 microscope (Leica, Wetzlar, Germany).

Mouse tumor, femurs, spleen, and lymph node were dissected and cleaned, followed by fixation in 10% formalin and paraffin embedding. Femurs were decalcified in Decalcifying Solution-Lite (Sigma, #D0818). For morphological analyses, tissue sections (4-μm) were prepared and stained with hematoxylin and eosin (H&E). The histological features of the tissues were observed and images captured using a light microscope (Olympus, Tokyo, Japan).

In a second experiment, 70 C57BL/6J, 35 CD1, and 35 Sv129Ev mice purchased from the respective vendor at 10 weeks of age and exposed to LCPS. Of these 70 C57BL/6J, 21 achieved dominant (all mice paired with a Sv129Ev) and 31 achieved subordinate social status (26/31 when paired with a CD1). Mice were sacrificed at 17 months of age for the collection of tissue specimens, at which time they were in apparent good health as this age precedes the rapid decline of the lifespan curve typical of this strain. These animals are not included in the survival analysis of the lifespan study. Mice were euthanized by CO₂ inhalation in the morning (between 8 and 10 a.m.). The dissection was performed as rapidly as possible following euthanasia. Major organs were removed, cut into appropriate size pieces, and either flash‐frozen in liquid nitrogen, or placed in 4% PFA for preservation. Flash‐frozen tissue samples were stored at −80°C. After several days of PFA fixation at room temperature, tissue fragments were transferred to 70% ethanol and stored at 4°C. Specimens of sternum were decalcified for 3 hr by “Decalcifying Solution‐Lite” (Sigma‐Aldrich, St. Louis, MO, USA). All dissectible tumors were analyzed and eight animals per group were randomly selected to conduct the senescence markers, heart morphology, and immunohistochemistry analyses as detailed below.