Histobond slides

Four hours after the onset of the restraint stress in Experiment 3, rats were euthanized with sodium pentobarbital (Fatal Plus, 0.2 ml i.v., Vortech Pharmaceuticals, Dearborn, MI, USA) and brains were extracted and flash-frozen on dry ice. The brains were serially sectioned at 30 µm for cannulae placement confirmation in the DMH (−2.6 mm to −3.8 mm bregma) using a cryostat (CM 1950, North Central Instruments, Plymouth, MN, USA) at −23 °C. Sections for cannulae placement confirmation were mounted on HistoBond slides (Cat No. 16004-406, VWR) in the cryostat and stained using cresyl violet to assist in the visualization of structures. Rats included in the analysis had confirmed bilateral cannulae placements in the DMH (Figure 2). The limits of the DMH were as defined by Fontes and colleagues (Fontes et al., 2011 (link)). Among sham-ADX rats, after confirming cannulae placements, eighteen of twenty-four rats were determined to be bilateral hits (sham-ADX/vehicle, n = 10; sham-ADX/CORT, n = 8).

Mice were injected i.p. with either vehicle (NaCl 0.9%) or insulin (0.75 U/kg body weight; Humalog) and 15 min after the injection, were anesthetized with isoflurane and perfused with 4 % paraformaldehyde. The livers were then explanted and kept overnight in paraformaldehyde. The samples were then transferred to a 30% sucrose solution to cryoprotect the tissue and thereafter the samples were imbedded in TissueTek and sectioned at 12 µm using a cryostat. Sections were mounted onto Histobond+ slides (VWR).
PIP₃ levels in the liver were determined using the Mouse on Mouse (M.O.M.) detection Kit following the manufacturer’s protocol (Vector Laboratories) as previously described^{19 (link)}. Samples were blocked with the MOM Mouse IgG blocking reagent for 1h and treated with MOM diluent for 5 min. The slides were then incubated for 30 min with the primary antibody (mouse anti-PIP₃; Echelon Biosciences Inc.) diluted 1:10 in MOM mouse diluent. Following this, samples were incubated for 10 min with the MOM biotinylated anti-mouse IgG solution and the secondary antibody (DyLight Streptavidin 488: Vector Laboratories) was added for 1h. Finally, slides were coverslipped with Vectashield and observed with a confocal microscope (Zeiss LSM700).