Biotinylated anti rabbit igg

Parenchymal IgG (BBB damage marker), Iba-1 (microglial/macrophage marker), and GFAP (astrocyte marker) immunohistochemistry were performed [16 (link)]. Briefly, 40-μm sections were incubated in 0.5% hydrogen peroxide in 0.1 M PBS (pH 7.4) containing 0.3% Triton-X100 (phosphate-buffered saline with triton-X100 (PBST)) for 30 min at room temperature (RT), washed with PBST, and blocked with PBST containing 2% bovine serum albumin for 30 min at RT. Sections were then incubated overnight at 4 °C with a rabbit anti-mouse IgG antibody (1:200 dilution; Jackson ImmunoResearch, West Grove, PA), rabbit antibody against Iba-1 (1:200 dilution, Wako Chemicals USA, Richmond, VA), or rabbit antibody against GFAP (1:2000 dilution, Abcam, Cambridge, MA). After washing with PBST, sections were incubated at RT for 1 h with biotinylated anti-rabbit IgG (1:500 dilution; Jackson ImmunoResearch, West Grove, PA), followed by 1 h incubation at RT with ABC complex, and developed with 3,3′-diaminobenzidine (DAB) (Vector Laboratories, Burlingame, CA) as per manufacturer’s instructions. Sixteen images per brain section were acquired at × 20 magnification, and the total positive immunoreactive area (expressed as percent of total analyzed area) was quantified using NIH Image J software 1.62 by an observer blinded to the experimental groups [16 (link)].

Immunohistochemistry was performed as previously described.28 In brief, serial 10‐μm‐thick sections were fixed with 4% PFA‐PBS for 10 minutes and blocked with Block Ace for 1 hour. The sections were incubated overnight with the following antibodies: phospho‐Akt (Ser473), phospho‐ERK (Thr202/Tyr204), and biotinylated Ki‐67 (#13‐5699, Thermo Fisher Scientific). They were reacted with biotinylated anti‐rabbit IgG (1:1000; #111‐065‐144, Jackson ImmunoResearch), except for anti‐Ki‐67 antibody‐treated sections, for 2 hours, followed by incubation with Elite ABC solution (#PK‐6100, Vector Laboratories) for 1 hour. After that, the sections were colored with 3,3′‐diaminobenzidine (DAB, #347‐00904, Dojin Chemicals, Kumamoto, Japan). Images were obtained using a BioZero microscope with a 4 × and 20 × Plan Apo objective lens.

The kidneys were fixed in formalin, embedded in paraffin and stained with PAS according to standard protocols. To analyse the expression of Ki-67, slides of fixed and paraffin-embedded mouse kidneys were de-paraffinized using Xylol and descending concentrations of ethanol. Antigen retrieval was carried out by warming kidney slides in citrate buffer (10 mM, pH6) for 10 min using a microwave. After blocking with 3% H₂O₂ and Avidin and Biotin (Vector Laboratories, Inc.) for 15 min each, slides were sequentially incubated with the Ki-67 antibody (rabbit Ki-67 ab16667, abcam, 1:500 dilution, over night at 4°C) and after washing with PBS with biotinylated anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA; 1 h at room temperature). Kidney slides were labelled with ABC kit (Vector Laboratories, Inc.), and development was carried out using diaminobenzidine solution (Sigma Aldrich). Slides were counterstained with hematoxylin (Sigma-Aldrich), dehydrated and afterwards mounted with Histomount (National Diagnostics). Stained slides were scanned using a Slidescanner (Leica) and analyzed using the ImageScope software (version 12.0.1.5030, Aperio).

For immunogold labeling, floating sections were treated for 30 min in 1% sodium borohydride in PB, to quench free aldehyde groups. The sections were incubated in 20% normal donkey serum (NDS) for 20 min to suppress non-specific binding, and then for 12 h in anti-Human Sept7 antibody (1∶300, IBL International GmbH, JP18991), along with 2% NDS. Sections were incubated in biotinylated anti-rabbit IgG (Jackson, USA) for 2 h. Following rinses in PBS, sections were incubated in streptavidin coupled to 1.4 nm gold particles (1∶100, Nanoprobes Inc.) for 2 h at room temperature, and rinsed in PBS. Sections were washed in 0.01 M sodium acetate (to remove phosphate and chloride ions), followed by silver enhancement with IntenSE M kit (Amersham Biosciences).
Sections for electron microscopy were postfixed in 0.5–1% osmium tetroxide in 0.1 M PBS for 35–45 min and stained en bloc with 1% uranyl acetate for 1 h. After dehydration in ascending ethanol series and propylene oxide, sections were infiltrated with Durcupan resin (Fluka) and flat-mounted between sheets of Aclar (EMS) within glass slides. Seventy nm sections were cut, mounted on 300 mesh copper grids, contrasted with Sato’s lead, and examined in a JEOL T1100 electron microscope at 80 KV; images were collected with a 12 bit 1024×1024 CCD camera.

Goat polyclonal anti-PCSK9 from R&D (Minneapolis, MN, United States), rabbit polyclonal anti-PCSK9 from Abclonal Science Inc. (Woburn, MA, United States), mouse monoclonal anti-IR-β subunit from Millipore (Etobicoke, ON, Canada), rabbit polyclonal anti-IR-α subunit from Biorbyt (Cambridge, United Kingdom); rabbit polyclonal anti LDL-R, rabbit polyclonal anti-ABCA1, purified rabbit polyclonal anti-SR-BI, rabbit polyclonal anti-SR-BII, rabbit polyclonal anti-IL-17 and rabbit polyclonal anti-LDL-R from Novus Biologicals (Littleton, CO, United States); rabbit polyclonal anti-ACAT-1 and anti-ACAT-2 from Cayman Chemical (Ann Arbor, MI); chicken polyclonal anti-hormone-sensitive lipase (HSL) from ProSci Inc. (Poway, CA, United States); and rabbit polyclonal anti-IL-17RA from Abcam (Cambridge, MA, United States); HRP-conjugated anti-mouse IgG, HRP-conjugated anti-rabbit IgG, HRP-conjugated anti-goat IgG, biotinylated anti-rabbit IgG from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, United States); HRP-conjugated anti-chicken IgG (H + L) made in goat from Vector Laboratories Inc. (Burlingame, CA, United States).