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 homogenates55 (link)–57 (link). In addition, downregulation of C1qbp expression using C1qbp-specific siRNA resulted in a reduced density band corresponding to C1qbp in Western blotting56 (link),57 (link).
The immunolabeling was performed as described before48 (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 immunolabeling was performed as described before48 (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.
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