Biotinylated goat anti mouse igg

To verify the presence of T. gondii cysts in brains of C. callosus, the samples were submitted to immunohistochemistry assay. For this purpose, the brains were fixed in 10% formalin in PBS for 24 h, embedded in paraffin and the glass slides containing sections of 4 μm obtained in microtome were treated for 5 min with citric acid (pH 6.0) for antigenic recovery. Next, 5% acetic acid solution was added on the sections for 8 min to avoid detection of endogenous phosphatase, and posteriorly the sections were treated with 2.5% goat serum for 45 min at 37°C to inhibit recognition of non-specific sites. After, the sections were incubated with 1:100 primary antibody (previously infected C. callosus serum) during 12 h at 4°C, washed in Tris-buffered saline solution (TBS, pH 7.4) to remove the excess of antibodies, and treated with 1:600 secondary antibody (biotinylated goat-anti mouse IgG, Jackson Immuno Research Laboratories, West Grove, PA, United States) for 1 h at 37°C. Finally, the detection of parasites was developed for 10 min with fast red naphthol (Sigma) at room temperature, and the brains counterstained with Harris’s hematoxylin were visualized under light microscope (BX40, Olympus, Tokyo, Japan) (Gomes et al., 2011 (link); Castro-Filice et al., 2014 (link)).

IHC was performed on paraffin-embedded sections. In brief, sections (5 μm) were deparaffinized and rehydrated. Primary antibodies were detected with biotinylated goat anti-mouse IgG (1:2000; Jackson ImmunoResearch Laboratories), biotinylated goat anti-mouse IgM (1:1500), or biotinylated goat anti-rabbit IgG (1:1800) (all from Jackson ImmunoResearch Laboratories) and visualized using an ABC reagent kit (Vector Laboratories, Burlingame, CA), according to the manufacturer’s recommendations. Bright-field images were acquired using a Carl Zeiss Axio Imager M2 microscope, equipped with an Axio Cam MRc5 color camera (Carl Zeiss, Oberkochen, Germany). Sections were counterstained with hematoxylin (Vector Laboratories) for nuclear staining. The following antibodies were used for immunostaining: rabbit anti-oligomer antibody A-11 (1:600), mouse anti-oligomer antibody F11G3 (1:100), and mouse anti-calbindin antibody (1:450).

Coronal sections of the brain were cut at a thickness of 30 µm using a vibrating microtome (vibratome 1000, the Vibratome Company, St. Louis, MO, USA). Primary antibodies, including mouse anti-tyrosine hydroxylase (TH; 1:2000; Sigma-Aldrich) or mouse anti-Parvalbumin (PV) (1:2000; Sigma-Aldrich), were used. Biotinylated goat anti-mouse IgG (1:500 the Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as the secondary antibody. Immunoreactivity was amplified using an avidin-biotin complex system (1:100, Vector Labs, Burlingame, CA, USA). TH is the key enzyme for dopamine synthesis, and TH immuno-histochemistry was used to label dopaminergic neurons. PV immunostaining was used to label PV-positive GABAergic interneurons. The densities of immunopositive cells were estimated using StereoInvestigator system (MicroBrightField Bioscience, Williston, VT, USA) [35 (link)].

Paraffin sections were processed as described above (see Immunofluorescence). Mouse monoclonal antibody to Cxd2 (AM392; BioGenex, San Ramon, CA, USA) and Isl1 antibody (40.2D6; Developmental Studies Hybridoma Bank, Iowa City, IA,USA) were incubated on sections overnight at 4°C. Sections were washed and incubated with a biotinylated goat anti-mouse IgG (115-065-146; Jackson ImmunoResearch) for 2 hours at room temperature. Slides were then washed and incubated for horseradish peroxidase-conjugated streptavidin (123-065-021; Jackson ImmunoResearch) for 2 hours at room temperature. Peroxidase activity was detected with the addition of diaminobenzidine (D4293; Sigma) and 0.1% H₂O₂. Sections were counterstained with hematoxylin, dehydrated, and covered with coverslips. Sections were photographed as described above (see Hematoxylin and eosin staining). See Additional file
2: Table S4 for details of specific immunohistochemistry protocols.

The immunohistochemical staining was performed with FAK 4.47 antibody (Millipore #05-537). For antigen retrieval, slides were heated in the microwave for 10 minutes in citrate buffer (pH 6.0), followed by a 15 minute cool period. Endogenous peroxidase was quenched with aqueous 3% H₂O₂ for 10 minutes and washed with 1×PBS with 0.5% Tween 20 solution. Slides were loaded on a DAKO autostainer and blocked with serum-free protein block solution (Dako #X0909) which was applied for 5 minutes and then FAK primary antibody (Millipore #05-537) was applied for one hour. The biotinylated goat anti-mouse IgG (Jackson Immuno Research Labs, #115-065-062) was applied for 30 minutes, followed by the Elite ABC Kit (Vectastain, #PK-6200) for 30 minutes, and the DAB chromagen (Dako, #K4007) for 5 minutes. The slides were counterstained with hematoxylin, dehydrated, cleared and cover slipped.