P1379 100ml

Cryosections were dried for 10 min at room temperature, fixed for 10 min in 10% formalin (#HT501128-4L, Sigma Aldrich, Germany). Embedding medium was removed with 0.02% Tween20 (T; #P1379-100ML, Sigma Aldrich, Germany) in tris buffered saline (TBS) twice for 5 min. All unspecific binding sites were blocked for 1 h at room temperature in 10% normal donkey serum (#S30-100ML, EMD Milipore, Germany), 1% bovine serum albumin (#0163.2, Roth, Germany) in TBST, followed by overnight incubation of the 1st antibody diluted as indicated in 1% bovine serum albumin in TBST at 4 °C. After 30 min at room temperature 1st antibody was carefully washed away with 4 repetitions of 5 min TBST. The 2nd antibody (A21247; A31571; A21434; A31572; A21432; Invitrogen and 703-545-155; Jackson ImmunoResearch) was diluted 1:500 in 1% bovine serum albumin in TBS, DAPI (300 nM, #D9542-5MG, Sigma Aldrich, Germany) was added, and after 1 h incubation at room temperature and 4 times 5 min wash in TBS, all sections were mounted with Dabco (#0718.1, Roth, Germany) in Mowiol (#0713.1 Roth, Germany). TUNEL staining (In Situ Cell Death Detection Kit, TMR red; #12156792910 Roche; Merck Germany) was applied according to the manual after 2nd antibody and before mounting with Dabco/Mowiol.

Fecal samples were collected to non-invasively measure levels of the stress hormone corticosterone via its metabolites, under baseline and stress-inducing conditions. Baseline samples were collected after mice were left unhandled for 48 h in their home cages, and stress samples were collected after exposure to predator odor in home cages overnight. Predator odor was prepared by sealing bedding sawdust (20 l) in a bin bag for 48 h with 10 squares of blotting paper (5 cm²) each infused with 50 μl of fox odor solution (50 μl TMT, 2,4,5-Trimethylthiazole 98%, 219185-5G, Sigma-Aldrich, United Kingdom), 420 μl water and 30 μl Tween-20 (P1379-100ML, Sigma-Aldrich, United Kingdom). For both baseline and stress-conditions mice were placed into empty individual cages from 9 am – 12 pm on the morning of sample collection, with fecal pellets being collected every 90 min and stored at −20°C until processing. FCMs were subsequently extracted and quantified with a 5α-pregnane-3β,11β,21-triol-20-one enzyme immunoassay, previously described in detail and fully validated for mice (Touma et al., 2003 (link), 2004 (link)). NB, due to the 8–12 h delay between a stress event and subsequent excretion of FCMs, separation of mice into individual unfamiliar cages would not affect the FCM levels of the baseline samples (Touma et al., 2003 (link), 2004 (link)).