Mice represents different housing conditions were euthanized (sodium pentobarbital 45mg/kg; intraperitonially) after the behavioural test. Hippocampus region (n = 6/group) was dissected out from the whole brain, and stored at -70°C. The hippocampus tissue from each individual was used to isolate total RNA, protein and Gas Chromatography-Mass Spectrometry (GC-MS) analysis, and feces (n = 6/group) also collected from the same individuals. The whole brain was dissected out (n = 4/group), and immediately processed for immunohistochemistry.
Hippocampus tissue was homogenized in lysis buffer [Tris-Hydrochloride (50mM; pH 7.5; Cat# RM613; HiMedia, India) Sodium chloride (150mM; Cat# 1.93206.0521; Merck, India), Ethylenediaminetetraactetic acid (EDTA) Disodium salt (50 mM; Cat# RM1195; HiMedia, India), Tergitol (NP-40; 0.1% V/V; Cat# MKCD6607; Sigma-Aldrich, India), Dithiotheritol (DTT,1 mM; Cat# MB070; HiMedia, India) Sodium orthovanadate (Na3Vo4, 0.2Mm; Cat# S6508; Sigma-Aldrich, India), Phenylmethylsulfonyl fluoride (PMSF;0.023mM Cat# P7626; Sigma-Aldrich, India)] following the procedure as reported [40 (link)]. Final supernatant was collected, concentration of each samples were measured using Bio-photometer Plus (Eppendorf Inc., Germany) and stored in aliquots at -80°C.
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