Goat anti rabbit

Immunohistochemistry was performed on formalin fixed celloidin embedded sections. The methodology for celloidin removal and antigen retrieval steps has been described in detail (Lopez et al., 2016 (link)). Tissue sections were incubated for one hour with a blocking solution containing 5% normal goat serum/1% bovine serum albumin (BSA) fraction-V (Sigma, St. Louis, MO) and 0.5% Triton X-100 (Sigma) in PBS. Incubation with primary antibodies against megalin and cubilin was performed for 48 hours at 4°C in a humid chamber. The sections were washed with PBS (3 ×15 minutes), and then incubated for one hour with either the goat anti-rabbit or the donkey anti-goat biotinylated secondary antibodies (1:1000, Vector Labs, Burlingame, CA), and then washed with PBS (3 × 15 minutes). Next, one-hour incubation was performed with Vectastain Elite ABC reagent (Vector Labs) followed by PBS washes (15 minutes × 3). Immunoperoxidase staining was performed using Immpact DAB solution (Vector Labs). The reaction was stopped with distilled water washes (15 minutes × 4). Slides were mounted with Vectamount AQ aqueous mounting media (Vector Labs) and glass coversliped.

After slices were incubated in primary antibody (Table 1) and washed, they were incubated for 3 hrs, at room temperature (RT) in biotinylated secondary antibody (1:600 goat anti-rabbit, Vector Laboratories, USA). The sections were placed in avidin-biotin peroxidase complex (ABC Elite, Vector Laboratories, USA) for 1 hr at RT. PBS was used after each step. Peroxidase activity was visualized as a brown precipitate by reacting the sections for 3–7 minutes at RT with a solution containing 0.04% 3–3diaminobenzidine-tetrachloride (DAB) and 0.006% H₂O₂. When picric acid was used for pre-staining tissue preservation, the DAB solution also included 0.4% nickel ammonium sulfate in 50 mM Tris-HCl buffer (pH 7.6) to yield a dark blue precipitate. The reaction was stopped by rinsing with PBS or Tris-HCl buffer, accordingly. For EM, the reaction product was intensified and substituted with silver/gold particles, as described by (Livneh et al. 2009 (link)). For control experiments, sections were processed as described above but without the primary antibody, resulting in no specific staining.
Sections for light microscopy were mounted on SuperFrost Plus slides (Menzel Glaser, Germany) and air-dried. The sections were then dehydrated in a graded ethanol series (70–100%), cleared with xylene, and cover-slipped with Enthellan for observation under an Olympus BX43 microscope.