Peroxidase conjugated goat anti mouse antibody

Western blot of neuronal protein extracts was performed as described previously [48 (link)]. The following primary antibodies were used: rabbit anti-Fbp (1:500, isolated and purified as described in [49 (link)]), rabbit anti-Camk2α (1:1000, Merck KGaA, Darmstadt, Germany, C6974) and mouse anti-β-actin (1:50,000, Merck KGaA, Darmstadt, Germany, A1978). The following secondary antibodies were used: goat anti-rabbit peroxidase-conjugated (1:50,000, Merck KGaA, Darmstadt, Germany, a0545) and goat anti-mouse peroxidase-conjugated antibodies (1:100,000, Merck KGaA, Darmstadt, Germany, a9044) were used. The reaction was visualized chemiluminescently.

Western blot of neuronal protein extracts was performed as described in [20 (link)] using the following antibodies: rabbit anti-Fbp (1:2000, [21 (link)]), mouse anti-Camk2α (1:1000, ThermoFisher Scientific, Waltham, MA, USA, MA1-048) and rabbit anti-tubulin β3 (1:1000, Synaptic System GmbH, Göttingen, Germany, 302302). Goat anti-rabbit peroxidase-conjugated (1:50,000, Merck KGaA, Darmstadt, Germany, a0545) and goat anti-mouse peroxidase-conjugated antibodies (1:50,000, Merck KGaA, Darmstadt, Germany, a9044) were used as secondary antibodies, and the reaction was visualized using the SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, Waltham, MA, USA).

Rats were anaesthetised with sodium pentobarbital (200 mg/kg) prior to transcardial perfusion with physiological saline for 3 min and 4% (wt/vol) paraformaldehyde in 0.1 m phosphate buffer for 10 min. The brains were removed and kept in 4% paraformaldehyde solution at 4 °C for at least 2 days before being transferred to cryoprotectant (30% wt/vol sucrose phosphate) for 2 days. Coronal sections (50 μm, 2 : 4) were cut with a base-sledge microtome. Half of the sections were stained immunohistochemically in situ with a monoclonal antibody for tyrosine hydroxylase (TH; Immunostar, Hudson, WI, USA; catalogue no. 22941; 1 : 4000 concentration), to check for DA-reactive terminals in both the NAc–DA and midbrain–DA lesion groups. Briefly, sections were washed and incubated for two nights in primary antibody and blocking serum (1% foetal calf serum, 5% normal goat serum and 0.2% Triton in PBS). Sections were then quenched with hydrogen peroxide and incubated for 1.5 h in secondary antibody (goat anti-mouse peroxidase-conjugated antibody; Sigma-Aldrich, Dorset, UK) and then developed with diaminobenzidine before being mounted, dehydrated and coverslipped. The rest of the sections were stained with Cresyl violet to assess the extent of the excitotoxic MFC lesions. Lesions are described in the nomenclature used by Paxinos & Watson (1998 ).