Avidin biotin peroxidase complex abc

After the microtomy, the sections of the first series were mounted on previously gelatinized, dried, and dehydrated slides. One week later, they were processed to delimit the cortical centers and subcortical nuclei of interest in the common marmoset monkey by the Nissl staining method and then covered with a cover glass. In the third series of cuts, dextran-BDA (with glucose-oxidase) was revealed; sections were first rinsed for 20 min in oxygen peroxide to quench endogenous peroxidase and subsequently incubated overnight under continuous shaking in avidin–biotin-peroxidase complex (ABC; Vector Laboratories, Burlingame, CA) + diaminobenzidine (DAB) + 0,1% glucose-oxidase (Sigma-Aldrich) + 1% ammonium nickel sulfate in 0.1 M phosphate buffer pH. 7.4. A week later, these tissues were subjected to the procedure of dehydration and diaphanization for intensification with Osmium. Finally, the other series were stored in an antifreeze solution for possible repetitions and data confirmation, as well as other neuronal study techniques.

Animals were anaesthetized with sodium pentobarbital (60 mg/kg, i.p.) and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffer (1 ml/g of body weight). Brains were removed and post-fixed for 2 h in the same solution, cryoprotected in 30% sucrose/phosphate buffer for 24 h and frozen in dry ice-cooled isopentane. Serial coronal sections (30 µm thick) through the whole brain were cut in a cryostat and collected in phosphate buffer. Free-floating sections were incubated for 15 min in H2O2 (0.3% phosphate buffer, 10% methanol). Thereafter, sections were incubated in a blocking solution (1% fetal bovine serum, 0.2 M glycine plus 0.5% Triton X100). After blocking with 10% normal serum and 0.2% bovine serum albumin, sections were rinsed and incubated overnight at 4°C using a monoclonal antibody against fibrillary acidic protein (GFAP, 1∶1000) (Dako, Denmark). Sections were washed and incubated with a biotinylated secondary antibody (1∶200 Sigma-Aldrich) for 2 h at room temperature. Afterwards sections were incubated with avidin-biotin-peroxidase complex (ABC; 1∶200; Vector, Burlingame, CA, USA). Peroxidase reaction was developed with 0.05% diaminobenzidine in 0.1 M phosphate buffer and 0.02% H2O2, and immunoreacted sections were mounted on gelatinized slides. Stained sections were examined under a light microscope (Olympus BX61).

The EF calcium-binding protein, Iba1, derived from the geneiba1 (ionized calcium binding adapter molecule 1) is a protein expressed only in microglia in the brain.⁷¹ (link) In mice, every third, while in rats, every fifth 40-μm-thick free-floating brain section of PLX3397-treated and control animals was processed for Iba1 immunohistochemistry. Sections from each experiment were pre-treated in PB containing 0.3% hydrogen peroxide (Sigma, St. Louis, Missouri, United States, Cat#: H1009) for 15 min for quenching of endogenous peroxidase activity. Then, the sections were incubated in PB containing 0.5% Triton X-100 (Sigma, Cat#: X100) and 3% bovine serum albumin (BSA; Sigma, Cat#: A3294) for 1 h. Sections were then incubated in rabbit anti-Iba1 antibody as a marker of microglial cells (1:1200; FUJIFILM Wako Shibayagi, Cat# 27030, RRID:AB_2314667) at room temperature for a night. Following the primary antibody, the sections were incubated in biotin-conjugated anti-rabbit IgG secondary antibody (1:800; Jackson ImmunoResearch, Cat#: 711-065-152) for 1 h and then in the avidin–biotin–peroxidase complex (ABC; 1:500; Vector Laboratories) for 1 h. The labeling was visualized by nickel-2’-diaminobenzidine (Ni-DAB) peroxidase technique. Sections were covered with Depex mounting medium (Sigma, Cat#:06522) after drying.

Free-floating coronal sections were rinsed in PBS, pH 7.35, and then treated with 0.5% H₂O₂ and 10% methanol in PBS for 15 minutes. After that, they were preincubated for 2 hours in a blocking solution (10% foetal bovine serum (FBS), 0.2 mM glycine, 0.1% Triton X-100 in 0.2% PBS-gelatine). Sections were then incubated overnight at 4 °C with the primary polyclonal antibody against calbindin (diluted 1:1000; Swant Inc, Switzerland).
Sections were then sequentially incubated with biotinylated secondary antibody (diluted 1:200; Sigma-Aldrich) for 2 hours at room temperature and then with the avidin-biotin-peroxidase complex (ABC; diluted 1:200; Vector Laboratories, USA). Peroxidase reactions were performed with 0.03% DAB in 0.1 M PB and 0.01% H₂O₂, and immunoreacted sections then mounted onto gelatinised slides. The stained sections were examined under a light microscope (Olympus, BX61, Olympus Optical, Hamburg, Germany).

Every fourth free-floating section of 5 rats was immunolabelled for C1qbp using a rabbit anti-C1qbp antibody (dilution 1:50, sc-48795, Santa Cruz Biotechnology Inc.). Western blotting using this antibody revealed a single band from cellular homogenates^{55 (link)–57 (link)}. In addition, downregulation of C1qbp expression using C1qbp-specific siRNA resulted in a reduced density band corresponding to C1qbp in Western blotting^{56 (link),57 (link)}.
The immunolabeling was performed as described before^{48 (link)}. Briefly, endogenous peroxidase activity was blocked with 0.9% H₂O₂. Non-specific binding sites were blocked with 3% bovine serum albumin (BSA), 0.5% Triton-X 100 and 0.05% sodium azide dissolved in PB for 1 h. After washing steps, the sections were incubated in biotin conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (1:1000, Jackson Immunoresearch, West Grove, PA) for 1 h and followed by the applying of avidin-biotin peroxidase complex (ABC; Vector Laboratories, Burlingame, CA, USA) for 1 hour. The labelling was visualized by incubation in the mixture of 0.02% 3,3-diaminobenzidine (DAB; Sigma), 0.08% nickel (II) sulphate, and 0.001% H₂O₂. After dehydration, sections were mounted on SuperFrost Ultra Plus Adhesive Slides coverslipped with DPX Mounting Medium.

The brain sections were incubated in 4% NGS for 30 min and then in a mixture of VGLUT1 (1:5000) and EndophilinA1 (1:500) antibodies, supplemented with 1% NGS overnight at RT. The sections were then incubated with goat anti-guinea-pig IgGs conjugated to biotin (1:200), washed in PBS and incubated in a mixture of avidin–biotin–peroxidase complex (ABC), (1:100; Vector Laboratories, Burlingame, CA) and goat anti-rabbit IgGs conjugated to gold particles (1.4 nm diameter; Nanoprobes, Stony Brook, N Y; 1:100 in PBS/BSA C) for 2 hr in PBS/BSA C. The sections were then washed in PBS and post-fixed in 1% glutaraldehyde in PBS. After washing (PBS; sodium acetate buffer, 0.1M, pH 7.0), the diameter of the gold immunoparticles was increased using a silver enhancement kit (HQ silver; Nanoprobes) for 5 min at RT in the dark. After washing (2xPBS, 1xTB 0.05M, pH 7.6), the immunoreactive sites VGLUT1 were revealed using DAB. The sections were then stored in PB and processed for electronmicroscopy.