Overall experimental design, brain samples, and tissue processing
Pilot experiments were carried out to assess sortilin immunolabeling with the two antibodies generated against the extracellular and intracellular C-terminal domains, respectively [
62]. These experiments used cryoprotected forebrain sections from transgenic mice available from earlier studies [
64,
65]. The animals were perfused at representative age time points before and after the onset of cerebral amyloid pathology: APP/PS1 mice at 2, 8, and 14 months; 5×FAD mice at 1, 2, and 6 months; and 3×Tg-AD mice at 4, 6, 12, 18, and 26 months. Overall, no plaque-like extracellular sortilin immunolabeling was observed in the processed sections. Therefore, for formal correlated anatomical/biochemical study, we obtained transgenic mice at selected age points: APP/PS1 mice at 14 months (
n = 8) (Better Biotechnology Co., Ltd., Nanjing, China); 5×FAD mice at 8 months (
n = 4) (The Jackson Laboratory, Bar Harbor, ME, USA), and 3×Tg-AD mice at 22 months (
n = 4) (in-house breeding, Southern Illinois University at Carbondale). The transgenic animals at the aforementioned age points have developed robust cerebral amyloid pathology as established earlier [
5,
6,
8,
64,
65]. Brain samples from C57BL/6 mice (6 months;
n = 8) were also used as negative assay controls.
Among the APP/PS1 and C57BL/6 mice, four animals were perfused transcardially under deep anesthesia (sodium pentobarbital 100 mg/kg intraperitoneally) with normal saline followed by 4% paraformaldehyde, with the brains dissected out, postfixed, and cryoprotected in 30% sucrose before sectioning at the frontal plane at 35-μm thickness in a cryostat. Other APP/PS1 (n = 4) and C57BL/6 mice (n = 4) were perfused with cold saline only, with the brains removed, snap-frozen, and stored at − 80 °C until tissue homogenization for immunoblot analysis. The 5×FAD and 3×Tg-AD mice were also perfused transcardially with cold saline first. After removal from the skull, brains were bisected along the cerebral sagittal fissure, with half brains immersed in 4% paraformaldehyde fixative for further histological processing as with the aforementioned perfusion-fixed mouse brains, whereas the other half brains were snap-frozen for Western blotting. To allow easy identification of sections from different transgenic strains after batch-processing immunohistochemistry, 5×FAD mouse hemibrain and 3×Tg-AD mouse hemibrain were sectioned (35 μm thick) along the frontal and sagittal planes, respectively. All mice were housed individually in a light-controlled (12-h/12-h on/off) and temperature-controlled (22–25 °C) vivarium with free access to food and water.
Monkey brain sections used in the experiments in the present study were available from earlier original studies (e.g., [
42,
66,
67]). Selection of the tissues/cases was based on pilot immunohistochemical assessment in a set of sections from each brain. Thus, cerebral sections used in the present study were from rhesus monkeys (
Macaca mulatta) at middle (
n = 2, 22, and 22.5 years old) and old (
n = 2, 27, and 30 years old) ages without cerebral amyloid pathology, as well as from four aged (30, 30.3, 31, and 34 years old) rhesus monkeys and three aged (29, 30.5, and 32 years old) cynomolgus monkeys (
Macaca fascicularis) with cerebral amyloid lesions. In the original studies, the monkeys were housed individually on a 12-h/12-h on/off lighting schedule with free access to food and water. They were sedated with ketamine (20 mg/kg intramuscularly) and anesthetized with sodium pentobarbital (25 mg/ kg intravenously) prior to transcardiac perfusion with normal saline followed by 4% paraformaldehyde. The brains were then removed and cryoprotected in 30% sucrose in 0.1 M sodium PBS at 4 °C. Serial sections (40 μm thick) across the cerebrum were cut frontally on a sliding frozen microtome and then stored at − 20 °C in cryoprotectant until use in the present study.
Postmortem human brains were obtained through the willed body donation program at Xiangya School of Medicine [
68]. After removal from the cranium, the cerebrum was bisected, with one hemisphere fresh-frozen and another hemisphere fixed by immersion in formalin followed by histological preparation. As part of standard brain banking protocol [
69], each brain was assessed for AD neuropathology with Aβ and tau immunolabeling in paraffin or cryostat sections from the temporal, prefrontal, and occipital lobes of the fixed hemisphere, with the extent of pathology (if present) scored according to Braak’s staging and the National Institutes of Health recommended guideline [
70‐
72]. For the present study, cryoprotected sections (40 μm thick) from the frontal and temporal lobes of the brains from aged cases (
n = 4) with an antemortem history of dementia (designated as AD cases) were used for immunohistochemical analysis. Temporal cortical samples from the frozen hemispheres of the same cases were obtained for Western blot analysis. These brains were taken with postmortem delays ≤ 18 h and showed Braak’s score of neurofibrillary tangle ≥ IV, with amyloid pathology present across all cortical lobes, including the hippocampal formation.
Animal use was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee of Central South University (mice) and Rush University (monkeys). Use of postmortem human brains was approved by the Ethics Committee for Research and Education at Xiangya School of Medicine, in compliance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).
Immunohistochemistry
Transgenic mouse, monkey, and human cerebral sections were processed in parallel for correlated examination of immunolabeling. Thus, three or four forebrain sections at different planes from one mouse, one prefrontal and one temporal lobe section from one monkey, and one frontal and one temporal lobe section from one human case, were batch-processed in each experiment for a given antibody. Adjacent sets of sections were immunohistochemically processed with the following primary antibodies: rabbit anti-sortilin intracellular C-terminal domain (1:2000, catalog number ab16640, raised against recombinant amino acids 800–831 of human sortilin, catalog number ab16686, Abcam Trading Shanghai Company Ltd., Shanghai, China), goat anti-sortilin extracellular domain (diluted at 1:2000, catalog number AF3154, raised against recombinant Gly76 to Asn753 of human sortilin, catalog number 2934-ST, R&D Systems China Co. Ltd., Shanghai, China), monoclonal mouse anti-Aβ 6E10 (1:4000, catalog number 39320, Signet Laboratories Inc., Dedham, MA, USA), rabbit anti-β-secretase (anti-BACE1) [
64,
65,
73], and rabbit anti-phosphorylated tau (1:4000, catalog number T6819, Sigma-Aldrich, St. Louis, MO, USA) or mouse anti-phosphorylated tau (PHF1, 1:4000; courtesy of Dr. P. Davis). Sections were first treated free-floating with 5% H
2O
2 in PBS for 30 min and 5% normal horse serum in PBS with 0.3% Triton X-100 for 1 h to lower nonspecific reactivity. For the sections subjected to 6E10 immunolabeling, an antigen retrieval step with formic acid treatment (1 h at room temperature) was applied prior to the aforementioned steps. Following incubation with the primary antibodies at 4 °C overnight, the sections were reacted with biotinylated pan-specific secondary antibody (horse anti-mouse, rabbit, and goat immunoglobulin G [IgG]) at 1:400 for 1 h and with avidin-biotin complex reagents (1:400; Vector Laboratories, Burlingame, CA, USA) for another 1 h. The immunoreactive product was visualized in 0.003% H
2O
2 and 0.05% 3,3′-diaminobenzidine, with sections mounted on microslides, dehydrated, and coverslipped for light microscopic examination.
Immunofluorescence
Double immunofluorescence was initiated with treatment of a batch of sections from transgenic mouse, monkey, and human cortex in PBS containing 5% donkey serum for 30 min. The sections were then incubated overnight at 4 °C with (1) mouse anti-Aβ antibody 6E10 (1:4000) and the rabbit anti-sortilin antibody (ab16640, 1:1000) or (2) 6E10 (1:4000) and rabbit anti-BACE1. Sections were then incubated at room temperature for 2 h with Alexa Fluor® 488-conjugated donkey anti-mouse IgG and Alexa Fluor® 594-conjugated donkey anti-rabbit IgG (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The monkey and human brain sections were treated with 0.1% Sudan Black to block autofluorescence after immunolabeling. All sections were counterstained with bisbenzimide (Hoechst 33342, 1:50,000, catalog number B2261, Sigma-Aldrich, St. Louis, MO, USA) and mounted with antifade medium before microscopic examination.
Western blot analysis
Frontal cortices were blocked from the frozen whole brains or hemibrains of the mice (n = 4/strain). Human cortical samples were blocked from the middle temporal lobes of the frozen hemispheres. Tissue samples were homogenized by sonication in Pierce T-PER extraction buffer (Thermo Fisher Scientific, Rockford, IL, USA) containing protease inhibitors (Roche, Indianapolis, IN, USA). Resulting brain lysates were centrifuged at 15,000 × g, with the supernatants collected and protein concentrations measured by DC detergent-compatible protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Extracts containing 50 μg of total protein were run in 10% or 15% sodium dodecyl sulfate-PAGE gels. Separated proteins were electrotransferred onto Trans-Blot pure nitrocellulose membranes (Bio-Rad Laboratories) and then immunoblotted with rabbit anti-sortilin (1:2000), mouse anti-Aβ 6E10 (1:4000), rabbit anti-phosphorylated tau (T6819, 1:2000), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000; Millipore Shanghai Trading Company Ltd., Shanghai, China) as loading controls. The membranes were further reacted with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:20,000; Bio-Rad Laboratories). Immunoblot signaling was visualized with the Pierce ECL-Plus Western Blotting Substrate detection kit (Thermo Fisher Scientific), followed by X-ray film exposure and image capture in a laser scanner.
Imaging, data analysis, and figure preparation
Immunolabeled sections were examined on an Olympus BX51 microscope (CellSens Standard; Olympus Corp., Tokyo, Japan) for basic assessment of histological integrity and immunolabeling. Light microscopic images were taken using a ×20 lens objective on a Motic Olympus microscope equipped with an automated stage and imaging system (Wuhan, China), which yielded non-edge-montaged and magnification-adjustable images covering the entire area of a glass slide. Immunofluorescent color images were captured on a Nikon confocal microscope using ×20 and ×40 lens objectives, with single-channel images extracted using the EZ-C1 FreeViewer version 3.70 software (Nikon, Tokyo, Japan). Immunoblot images were densitometrically analyzed using OptiQuant software (Packard Instrument Co., Meriden, CT, USA). Optical densities over target protein bands were measured with the rectangular selection tool, exported into Excel spreadsheets, and rearranged according to groups, with relative density levels calculated against internal references. Data were then entered into Prism software spreadsheets and graphed (GraphPad Software, La Jolla, CA, USA). Statistical analyses were carried out using a nonparametric test (Kruskal-Wallis test with post hoc Dunn’s multiple comparison using Prism 4.1 software), with the level of statistical significance set at p < 0.05. Figures were assembled using Photoshop 7.1 (Adobe Systems, San Jose, CA, USA).