Cells, media, and plasmids
Saccharomyces cerevisiae strain EBY100 was used for VLR surface display. The plasmid used for VLR library cloning and display was pCT-ESO75. VLR RBC3631 which specifically recognizes the human blood group type II H trisaccharide (Fucα1,2-Galβ1,4-GlcNAc) was used as an isotype control where indicated. For all yeast surface display experiments, EBY100 yeast were first grown overnight at 30 °C 260 rpm in SD-CAA media (20 g/L dextrose, 6.7 g/L yeast nitrogen base, 100 mM sodium phosphate buffer pH 6.0, 5.0 g/L bacto-casamino acids without tryptophan and uracil). The day before an experiment all yeast cultures were re-set to an OD600 of ~ 0.4 and grown for 3–4 h until reaching an OD600 of 1. Then, surface display was induced via switching to SG-CAA induction media (same recipe as SD-CAA except galactose is used instead of dextrose) and cultures were grown at 20 °C, 260 rpm for 16–18 h. HEK293F cells were purchased from ATCC (CRL-1573) and maintained in Freestyle F17 Medium (Thermo Fisher) at 37 °C, 8% CO2, and 135 rpm in a humidified incubator. The plasmid used for production of soluble VLR-Fc was pIRES-VLR-Fc. bEnd.3 cells at passage 22 were purchased from ATCC (CRL-2299) and maintained in complete growth media (DMEM supplemented with 4 mM l-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, 1500 mg/L sodium bicarbonate, and10% fetal bovine serum) at 37 °C, and 5%CO2 in a humidified incubator up to passage 30.
Animals
Male C57BL/6 mice (Mus musculus) at 6 to 7 weeks of age were purchased from Envigo and used in terminal experiments. All mouse experiments were approved by the UW-Madison Institutional Animal Care and Use Committee (IACUC) and performed in compliance with the UW-Madison IACUC and following National Institutes of Health (NIH) guidelines for care and use of laboratory animals. Sea lamprey larvae (Petromyzon marinus) captured from the wild by commercial fishermen (Lamprey Services, Ludington, MI) were maintained in sand-lined, aerated aquariums at 16–20 °C and fed brewer’s yeast. All lamprey experiments were approved by the Emory University IACUC and performed in compliance with the Emory University IACUC and following National Institutes of Health (NIH) guidelines for care and use of laboratory animals. All experiments and data analysis were performed in accordance with the ARRIVE guidelines.
Human tissue
De-identified normal human brain tissue was obtained from surgeries for other indications. All research was carried out in compliance and under the supervision of the University of Wisconsin-Madison Institutional Review Board and following the guidelines of the federal Common Rule. Patients give informed consent for surgery at the University of Wisconsin Hospital including a consent provision for the research use of leftover tissue removed during surgery.
Capillary isolation, plasma membrane fractionation, and quality analysis
Brains were removed from 6–7 week old male C57BL/6 mice (~ 20 g) and stored in DMEM on ice. Microvessels were isolated and endothelial plasma membranes fractionated essentially as previously described40. Briefly, the cerebellum and white matter were dissected away and brains were rolled on Whatman 3MM chromatography blotting paper to remove the meninges. Up to 15 brains were homogenized in 20 mL DMEM + 0.2%BSA in a dounce homogenizer, and the homogenate was passed over a 150 μm nylon mesh to remove large debris. The homogenate was mixed with an equal volume of 40% dextran solution and centrifuged at 5000×g for 15 min at 4 °C. The supernatant was discarded, and the crude microvessel pellet was resuspended in DMEM + 0.2% BSA. Microvessels were then recovered on 41 μm nylon mesh filters and washed twice with PBS. To prepare biotinylated plasma membrane proteins for yeast display library screening, microvessel membrane proteins were biotinylated prior to plasma membrane fractionation via incubation with 5 mM sulfo-NHS-LC-biotin (Thermo Fisher), which is membrane impermeable, for up to 2 h at 4 °C. Unreacted biotinylation reagent was quenched by addition of glycine to a final concentration of 100 mM and 10 min incubation on ice. Biotinylated microvessels were washed twice with PBS + 100 mM glycine to ensure complete quenching and removal of unreacted biotinylation reagent. Plasma membranes prepared for lamprey immunization were not biotinylated. Endothelial plasma membranes were fractionated from the purified microvessels via a two-step hypotonic lysis: (1) distilled water at 4 °C for 2 h and (2) 10 mM Tris–HCl pH 7.4 at 4 °C for 30 min. This was followed by sonication in 50 mM Tris–HCl pH 7.4 and centrifugation at 25,000×g. This resulted in a supernatant containing dispersed plasma membrane fragments and a pellet containing the capillary basement membranes. The supernatant fraction is referred to as brain capillary plasma membranes (BMPM) and used for lamprey immunization and yeast display screening. All buffers contained protease inhibitor cocktail (PIC, Roche, 11836170001) and 2 mM EDTA. Total protein concentration in all fractions was quantified using a BCA assay kit (Thermo Fisher) following the manufacturer’s instructions. This isolation procedure yielded 255 ± 35 μg of BMPM proteins from 15 mice. For quality analysis of the plasma membrane fractionation via western blotting, 10 µg of total protein from each fraction was separated via SDS-PAGE and transferred to nitrocellulose. Western blotting for brain capillary endothelial membrane marker Glut1 was carried out using a 1:1000 diluted rabbit anti-Glut1 (Thermo Fisher, PA1-46152). Western blotting for astrocyte endfoot marker GFAP (astrocyte endfeet are tightly associated with the basement membrane) was achieved with a 1:1000 dilution of mouse-anti-GFAP (BD Biosciences, 556329). Further quality analysis was achieved via γ-glutamyl-transpeptidase (GGT) activity assay as previously described76.
Lamprey immunizations
Sea lamprey larvae were sedated with 0.1 g/L tricainemethanesulfonate (Tricaine-S; Western Chemical, Inc.), then injected into the coelomic cavity with 50 µg of BMPMs in 30 µl of PBS. Three lampreys were immunized a total of three times at two week intervals and blood was collected two weeks after the final immunization from lampreys euthanized with 1 g/L Tricaine-S. Approximately 200 µl of blood was collected in 200 µl of PBS containing 30 mM EDTA as an anticoagulant. Blood plasma and leukocytes were separated from erythrocytes by layering the blood on top of 55% Percoll and centrifugation at 400×g for 5 min. Erythrocytes pelleted to the bottom of the tube, while leukocytes collected at the 55% Percoll interface and plasma remained above the interface. Buffer was added to the plasma samples to a final concentration of 20 mM MOPS/0.025% sodium azide pH 7.5 and stored at 4 °C. Leukocytes were stored in RNAlater at − 80 °C until needed for VLRB cDNA library cloning.
VLR library cloning
RNA isolated from total leukocytes using the Qiagen RNeasy kit was reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen) and oligo-dT priming. VLRB transcripts were amplified from the leukocyte cDNA by nested PCR using KOD high-fidelity DNA polymerase (Novagen). The first round of PCR utilized primers to the 5′ and 3′ untranslated region, (5′-CTCCGCTACTCGGCCTGCA) and (5′-CCGCCATCCCCGACCTTTG), respectively. The second round of PCR used primers that amplified only the VLRB antigen-binding domain from the LRRNT (5′-GCATGTCCCTCGCAGTG) to the LRRCT (5′-CGTGGTCGTAGCAACGTAG), and 50 bp of sequence homology to the yeast surface display vector was added to each primer for cloning by in vivo homologous recombination in transfected yeast cells. PCR products were excised from 1% agarose gels, purified using the Promega Wizard gel extraction kit and eluted in water. The pCT-ESO-BDNF yeast surface expression plasmid was digested with NheI, BamHI and NcoI to linearize the vector and remove the BDNF insert. Prior to transformation with the VLR library, yeast were grown to log-phase in SD-CAA media 30 °C until the culture density reached ~ 1 OD600. The yeast cells were harvested by centrifugation at 1000xg, washed in Milli-Q water, then incubated in 10 mM Tris/10 mM DTT/100 mM LiOAC, pH 7.6 at 225 rpm 30 °C for 20 min. After the incubation, the yeast cells were washed in Milli-Q water and resuspended in 1 M sorbitol at 1 × 109 cells/ml. 200 µl of yeast cells were mixed with 1 µg of digested vector and 2 µg of the purified VLRB PCR product and added to a 0.2 cm electroporation cuvette on ice. The yeast were electroporated at 2.5 kV (12.5 kV/cm) using a Biorad Micropulser. After electroporation, the yeast cells were incubated in a 1:1 mixture of 1 M sorbitol and YPD media (Fisher Scientific) at 30 °C for 1 h, then transferred to SD-CAA media. A small aliquot of the electroporated yeast cells was serially diluted in SD-CAA media and plated on SD-CAA agar plates to calculate the total number of transformants. Three electroporated samples were combined resulting in a library of 7.5 × 106 VLR clones. Aliquots of the yeast library were stored at − 80 °C in 15% glycerol.
YSD library screening with detergent solubilized BMPM proteins
VLR display libraries and control yeast displaying VLR-RBC36 were grown and induced as described above for each round of YSD screening. Two rounds of screening via the YDIP method were carried out as previously described42 with modifications. In each round, ~ 250 µg freshly isolated biotinylated BMPM proteins were solubilized in a final volume of 1 mL PBS containing protease inhibitor cocktail (Roche), 2 mM EDTA, 1 mM Biotin, 1% w/v BSA, and 1% v/v TritonX-100. To ensure complete solubilization of membrane proteins the mixture was incubated for 15 min at 4 °C and insoluble debris was removed via centrifugation. The first round of screening was carried out using a magnetic activated cell sorting (MACS) protocol77 to recover VLR binding to biotinylated BMPM antigens. Briefly, 2.1 × 108 yeast, 30-fold excess of starting library size, were incubated with 1 mL detergent solubilized BMPMs for 2 h at 4 °C with rotation. Yeast were then washed twice with 1 mL ice cold PBS + 1% TX-100 + 1%BSA (PBSTXA) and once with ice cold PBS + 1% BSA (PBSA). Washed yeast were resuspended in 0.5 mL ice cold PBSA, then 50 µL streptavidin microbeads (Miltenyi, 130-048-102) were added, and the mixture was incubated at 4 °C with rotation for 30 min. Microbead-bound yeast were washed once with 1 mL PBSA and resuspended in 0.5 mL PBSA. The 0.5 mL microbead-yeast suspension was applied to an LS column (Miltenyi, 130-042-401) placed within a Midi-MACS separator magnet (Miltenyi, 130-042-302). The column was washed twice with 3 mL ice cold PBSA, removed from the magnet, and yeast were eluted via plunging with 3 mL SD-CAA media. Dilutions of the eluate were plated to count the number of yeast recovered and the remaining yeast regrown for subsequent screening. In the second round of screening fluorescent activated cell sorting (FACS) was employed to further enrich for BMPM binders. 5 × 107 yeast were incubated with 0.5 mL detergent solubilized BMPMs for two hours at 4 °C with rotation. Full length VLR expression was detected via labeling with rabbit-anti-cmyc epitope (Thermo Fisher, PA1-981) followed by a goat-anti-Rabbit IgG-Alexa488 secondary (Thermo Fisher, A-11008). Binding to biotinylated BMPM antigens was detected by labeling with a mouse-anti-biotin (Labvison, BTN.4) followed by a goat-anti-mouse IgG-allophycocyanin (Thermo Fisher, A-865). 3 × 107 labeled yeast were sorted on a Becton Dickson SORP FACSAriaII (University of Wisconsin Carbone Cancer Center) to recover yeast double positive for VLR expression and BMPM antigen binding, and the sorted yeast were expanded in SD-CAA (Fig. S10).
YSD library biopanning
A two-step biopanning method was developed and applied to remove extracellular matrix (ECM) binding VLRs from the FACS-sorted library while enriching for VLRs that bind to extracellular epitopes using the bEnd.3 MBEC line. For each round, two substrates were used for biopanning. One 6-well plate containing decellularized ECM from bEnd.3 culture was prepared by growing cells to ~ 90% confluence then switching the cells to media supplemented with 5% ~ 500 kDa dextran sulfate (DxS, Acros Organics, 433240050) to promote robust ECM deposition78. After 4–6 days in D × S, cells were washed with PBS and plates were decellularized via a non-enzymatic protocol to leave behind intact ECM79,80. This plate was used in the ECM subtraction step. A second plate containing bEnd.3 cells grown to confluence under normal culture conditions was also prepared and used for the MBEC binding step. Prior to incubation with yeast both ECM and MBEC, plates were blocked for 30 min with PBSA at 4 °C. ECM subtraction was initiated by addition of induced yeast libraries or control yeast expressing VLR RBC36 suspended in PBSA into wells of the ECM plate at a density of ~ 0.85 × 106 yeast/cm2. The plate was incubated with gentle rocking for 2 h at 4 °C. Non-binding yeast were recovered from the ECM subtracted plate after 2 washes with ice cold PBSA and immediately applied to the MBEC binding plate for 2 h at 4 °C with gentle rocking. Non-binding yeast were removed by 3 washes with ice cold PBSA, and MBEC binding yeast were then recovered by scraping the cells into SD-CAA media. Dilutions of the MBEC binding cells were plated to count the number of yeast recovered and the remainder were expanded for subsequent rounds of biopanning or individual clone analysis.
VLR-Fc subcloning, production, and purification
VLR identified from the YSD screen were cloned into an expression vector, pIRES-VLR-Fc constructed from pIRESpuro2 (Clontech, 6937-1), by cloning rabbit IgG-Fc into the AgeI and BamHI sites. The expression vector included the VLRB signal peptide upstream of the multiple cloning site (MCS) to promote secretion of VLR into the culture media and the rabbit IgG-Fc downstream of the MCS to enable simple purification of VLR-Fc fusion proteins via ProteinA/G chromatography. VLR sequences were amplified via PCR with the following primers: VLRB-NT-NheI-F (5′-GAGAGCTAGCTGTCCCTCGCAGTGTTCG) and VLRB-CT-AgeI-R (5′-GAGAACCGGTCGTGGTCGTAGCAACGTAG). PCR products were digested with NheI and AgeI and ligated into the pIRES-VLR-Fc vector.
Soluble VLR-Fc fusion proteins were expressed by transient transfection of HEK293F suspension cultures. 80 µg pIRES-VLR-Fc plasmid DNA was mixed with 160 µg PEI (Polysciences, 23966) in 3 mL OptiPRO SFM (Thermo Fisher, 12309019) for 15 min and then applied dropwise to 80 mL HEK293F cultures. Transfected cultures were then incubated for 5–7 days at 37 °C, 8% CO2, 135 rpm in a humidified incubator and the supernatant containing secreted VLR-Fc was recovered via centrifugation and filtration. VLR-Fcs were purified from the cleared supernatant via gravity-driven chromatography over a packed bed of 100 µL Protein A/G Plus Agarose beads (Thermo Fisher, PI20423). After washing, three 200 µl fractions were eluted from the column with 100 mM Citric Acid pH 3 and neutralized with 1 M Tris-base pH 9, which typically yielded ~ 0.5 mg purified proteins from an 80 mL transfected culture. Purified proteins were stored for up to 2 months at 4 °C.
Immunolabeling of tissue and cells with VLR-Fc
Fourteen micrometer coronal brain cryosections from male C57BL/6 mice were washed in PBS and then blocked and permeabilized with immunolabeling buffer (PBS + 10% goat serum + 1% BSA + 0.05% saponin) for 30 min at room temperature. Next, purified VLR-Fcs at 5 µg/mL in labeling buffer were incubated on the brain slices for 1–2 h at room temperature. After washing, brain sections were incubated with Goat-anti-Rabbit IgG-Alexa555 secondary to detect VLR-Fc binding and Isolectin B4-Alexa488 (Thermo Fisher, I21411) as a brain microvessel marker for 1 h on ice. After washing, sections were post-fixed with 4% PFA, nuclei labeled with DAPI, and mounted in ProLong Gold antifade reagent (Thermo Fisher, P10144). Cell surface binding on live bEnd.3 cells was carried out by incubation with 5 µg/mL purified VLR-Fc proteins in PBS + 10% Goat serum + 1%BSA (PBSGA) for 1 h at 4 °C. After washing VLR-Fc binding was detected by staining with Goat-anti-Rabbit IgG-Alexa555 in PBSGA for 30 min on ice. After washing, cells were post-fixed with 4% PFA, nuclei labeled with DAPI, and mounted in ProLong Gold antifade reagent. For whole cell labeling, cells were prefixed with 2% PFA, then blocked and permeabilized in immunolabeling buffer prior to incubation with VLR-Fc and detection reagents as described for cell surface binding. In all cases images were obtained with a Zeiss Imager Z2 Microscope equipped with an AxioCam MRm using 10 × or 63 × objectives. Human brain samples were obtained with approval from the University of Wisconsin-Madison Institutional Review Board, sectioned and labeled via the methods described above for mouse sections.
Co-labeling VLR-30-Fc and NeuN was done on 8 µm mouse brain cryosections labeled that had been transcardially perfused with DyLight488 conjugated tomato lectin (LEL, Vector Laboratories, DL-1174). Sections were labeled with VLR-Fc-30 as described above, with the exception that for this assay a VLR-human Fc fusion protein was used, and consequently the secondary antibody was a Goat-anti-Human IgG-Alexa 555. Following labeling and staining for VLR-Fc, sections were incubated with anti-NeuN antibody (Abcam, ab104225) overnight at 4 °C in PBSGA, the sections were then washed and labeled with Goat-anti-Rabbit IgG-Alexa 647 30 min in PBSGA before washing and mounting in ProLong Gold antifade reagent.
Cell-based assays
Internalization assays– bEnd.3 cells analyzed by immunofluorescence microscopy were grown to confluence on glass coverslips. bEnd.3 cells used in quantitative internalization assays were grown to confluence in 96-well flat-bottomed plates (Corning, 353948). Cells were serum starved for 1 h at 37 °C in serum-free complete growth media. For endocytosis inhibitor experiments, 10 µg/mL (15.3 µM) fillipin, 20 µg/mL (62.7 µM) chlorpromazine, or 250 µg/mL (940 µM) amiloride were pre-incubated with the cells, transferrin-555 and cholera toxin subunit B-555 were used as controls. Subsequently, purified VLR-Fc diluted in serum free complete growth media were applied to the cells. Conditions were varied depending on the experiment. For temperature dependent internalization assays one group of cells was incubated with 10 µg/mL VLR-Fc at 37 °C and one group with the same concentration of VLR-Fc at 4 °C. Both groups were incubated for 30 min prior to subsequent labeling steps. For saturation experiments, all cells were incubated for 20 min at 37 °C with varying concentrations of VLR-Fc up to 4 µM. Samples for microscopy analysis were processed as follows. After the VLR-Fc incubation period, bEnd.3 cells were washed 3 × with ice cold PBS and incubated with Goat-anti-Rabbit IgG-Alexa488 in PBSGA for 30 min on ice to label cell surface bound VLR-Fc. Following washes, cells were fixed in 2% PFA for 10 min at room temperature and then blocked and permeabilized in immunolabeling buffer for 30 min on ice. To differentially label internalized VLR-Fc the fixed and permeabilized cells were incubated with Goat-anti-Rabbit IgG-Alexa555 in immunolabeling buffer for 30 min on ice. After washing, cells were post-fixed with 4% PFA, nuclei labeled with DAPI, and mounted in ProLong Gold Antifade Reagent. Samples were analyzed via widefield and/or confocal microscopy as described below. Quantification of the internalization ratio was done using ImageJ, the ratio of the total image intensity for the 555 internal VLR-Fc to 488 external VLR-Fc images was calculated and normalized to the control well for each VLR-Fc. Similarly, for the transferrin and cholera toxin controls, the total image intensity of the 555 channel was normalized to the control without inhibitor.
Samples for quantitative analysis of internalized VLR-Fc for temperature-dependent internalization measurements were processed as follows. bEnd.3 cells were first acid washed by 5 changes of ice-cold 0.9% w/v saline, pH 2.5 for a total of 25 min to remove cell-surface bound VLR-Fc. This stripping procedure routinely resulted in the removal of ~ 90% of the cell-surface bound VLR-Fc signal (Fig. S12). Cells were then fixed with 2% PFA and blocked and permeabilized in Odyssey Blocking Buffer (Li-Cor, 927-40000) + 0.1% TX-100 for 30 min at room temperature. Internalized VLR-Fc were detected by IRdye800CW Goat-anti-rabbit IgG (Li-Cor, 925-32211) and cell number in each well estimated with CellTag 700 (Li-Cor, 926-041090) both diluted in Odyssey Blocking Buffer and incubated with cells for 1 h at room temperature. After extensive washes with ice cold PBS + 0.1% Tween-20 and drying of the plate, signal in each well was measured with a Li-Cor Odyssey Imager with a focus offset of 3 mm and resolution of 169 µm. VLR-Fc signal in each well was normalized to a per cell basis via dividing by the CellTag 700 signal.
Equilibrium Binding Measurements-bEnd.3 cells were grown to confluence in 96-well flat-bottomed plates, washed 3X in PBS, and fixed with 2% PFA for 10 min at room temperature. Fixed cells were blocked and permeabilized as described above. Equilibrium affinity titration measurements were achieved via incubation of the cells with purified VLR-Fc diluted to a range of concentrations from 800 pM to 4 µM at room temperature for 2 h. After extensive washing with ice cold PBS + 0.1% Tween-20 cells were labeled for detection with the IRDye reagents and analyzed as described above. Fraction of cellular antigen sites bound by VLR-Fc was quantified using background subtracted per-cell binding signal and the data was fit to a bimolecular equilibrium binding model to determine the dissociation constant (KD).
Competition assay—recombinant receptor ecto-domain proteins (2 μM), rIR (R&D systems, 7544-MR), rLDLR (R&D systems, 2255-LD), and rTfR (Sino Biologics, 50741-M07H) were incubated with 200 nM VLR-Fc proteins in serum free complete growth media for 30 min and then applied to serum starved bEnd.3 cells in 96-well plates to allow for VLR-Fc binding to cell surface receptors. Plates were incubated at 4 °C for 2 h. After extensive washing with ice cold PBS cells were fixed with 2% PFA, permeabilized, labeled with IRDye reagents, and analyzed as described above.
Sialidase pre-treatment assay- bEND.3 cells were grown to confluence in 96-well flat-bottom plate. Cells were fixed in 4% PFA for 20 min at room temperature, washed in PBS, then blocked in Odyssey Blocking Buffer for 90 min at room temperature. In some cases, cells were pre-incubated with sialidase (P0720L, New England Biolabs) for 30 min at 37 °C to cleave glycans containing terminal sialic acid motifs. VLR-Fc (15 μg/mL) or biotinylated lectin (15 μg/mL) were added to the cells and incubated for 2 h at 4 °C to allow for binding. Lectins used were SNA (sambucus niagra agglutinin, Vector Labs, B-1305-2), MALII (Maackia Amurensis Lectin II, Vector Labs, B-1265-1), and ConA (Concanavalin A, Vector Labs, B-1005-5). After washing with ice cold PBS + 0.1% Tween-20, VLR-Fc were labeled with IRdye800CW Goat anti-Rabbit IgG (925-32211, Li-Cor), lectin with IRdye800CW Streptavidin (925-32230, Li-Cor), and cell number with CellTag 700 (926-041090, Li-Cor). The plate was washed with ice cold PBS + 0.1%Tween-20 and dried. Signal was detected with a Li-Cor Odyssey Imager with a focus offset of 3 mm and resolution of 169 μm. Binding signal in each well was normalized to cell number using the CellTag700 signal.
In vivo VLR-Fc brain targeting experiments
Male C57BL/6 mice (~ 20 g) were injected intravenously with 10 mg/kg VLR-Fc or positive control anti-TfR (8D3, AbDSerotec) in PBS. 8D3 was used as a positive control to validate internalization of the VLR-Fcs. After 1 h of antibody circulation mice were deeply anesthetized and the thoracic cavity was opened and transcardial perfusion was initiated via insertion of a catheter into the left ventricle and clipping of the right atrium. Ice cold wash buffer containing Earle’s balanced salts, 20 mM HEPES, 1 g/L glucose, 10 g/L BSA, and 5 mg/L DyLight488 conjugated tomato lectin (LEL, Vector Laboratories, DL-1174) was perfused at 5 mL/min for 5 min with a peristaltic pump to wash away unbound antibodies and label the vessel lumen with lectin. Then perfusion fixation with room temperature 4% PFA was carried out at the same flowrate for 10 min. Upon completion of perfusion the brain, heart, liver, and kidneys were dissected, and flash frozen in liquid nitrogen or stored in ice cold PBS. For immunofluorescence analysis, brains were cryopreserved in OCT and stored at − 80 °C prior to sectioning. For electron microscopic analysis tissue was immediately cut into 150 μm thick coronal sections on a vibratome and stored in fixative containing 4% PFA and 0.01% glutaraldehyde overnight at 4 °C with gentle agitation.
Sample preparation and immunofluorescence microscopy
Thirty or eight micrometer thick coronal brain sections were cut on a cryostat, and adhered to positively charged glass slides. Sections were washed with PBS to remove embedding compound and fixed with 2% PFA. Tissue was blocked and permeabilized in immunolabeling buffer for 30 min at room temperature. To visualize VLR-Fc in the brain sections, tissue was incubated overnight with goat-anti-rabbit IgG Alexa555 in immunolabeling buffer at 4 °C. In some cases, a goat-anti-collagen IV antibody (AB769, EMD Millipore) diluted in donkey immunolabeling buffer (goat serum replaced by donkey serum) was incubated on the sections for 2 h at 4 °C. Subsequently, sections were incubated with donkey-anti-rabbit IgG-Alexa555 conjugate and donkey-anti-goat IgG-Alexa647 conjugate in donkey immunolabeling buffer overnight at 4 °C. In all cases, sections were post-fixed in 4% PFA, nuclei labeled with DAPI, and mounted in ProLong Gold antifade reagent. Low magnification widefield images were obtained on a Zeiss Imager Z2 Microscope equipped with an AxioCam MRm using a 10 × objective, 100 × images were taken on an Olympus Ix70 microscope equipped with a Hamamatsu ORCA-flash4.0LT camera. Confocal imaging was performed on a NikonAR1 microscope using a Plan Apo λ 60 × oil objective with 1.4 numerical aperture and optical z-sections were obtained with a step size of 250 nm. Z-stacks were typically taken through a thickness of 5–10 μm. Images were 12-bit, 1024 × 1024 pixels, with a pixel size of 100, 110, or 120 nm. Maximum intensity projections of the Z-stacks were created using the Maximum Intensity Projection tool in NIS Elements (Nikon Metrology). In some cases, image contrast and brightness was adjusted for clarity of presentation using ImageJ. In these cases, all related images and controls were processed in an identical manner.
Quantification of vascular biodistribution was done using ImageJ. First, images were adjusted to threshold VLR-Fc-RBC36 negative control parenchymal binding and identical adjustment was subsequently performed for each VLR image. Regions of interest corresponding with positive lectin staining were outlined and the average fluorescence intensity corresponding to VLR-Fc vascular binding was measured. The background parenchymal intensity was also measured as an average of three small random regions within the parenchyma. A ratio of these values was used to determine relative vascular binding. The same quantification method was used for the naïve and IV injected tissues.
Quantification of postvascular accumulation of VLR-Fc-30 was done by counting the number of nuclei in each 8 μm brain tissue section field of view, assigning puncta to the nearest nuclei and counting the number of nuclei in each field of view with associated puncta, or counting those cells which showed a strong, whole cell labeling. This was performed on both VLR-Fc-30 and VLR-Fc-RBC36 injected animal tissue.
Sample preparation and electron microscopy
Immunogold labeling of 150 μm vibratome sections from mice injected with VLR-Fc was carried out with reagents purchased from Electron Microscopy Sciences (EMS) essentially following the manufacturers protocols. After aldehyde quenching with 0.1% NaBH4, sections were permeabilized via incubation with 0.1% TritonX-100 in PBS for 30 min, and then blocked with AURION Goat Serum Blocking Solution (EMS, 25596) for 1–2 h at room temperature. Subsequently, sections were incubated with Goat-anti-rabbit IgG-Ultrasmall Gold conjugate (EMS, 25100) diluted in PBS + 0.2% AURION BSA-c (EMS, 25557) overnight at 4 °C with gentle agitation. Following extensive washing sections were fixed in 2% glutaraldehyde for 30 min. Silver enhancement was carried out using the R-Gent silver enhancement kit (EMS, 25520) following the manufacturer’s instructions to increase the size of the ultrasmall gold particles. Sections were post fixed in 0.5% Osmium Tetroxide, 1% potassium ferrocyanide in 0.1 M sodium phosphate buffer for 1 h at room temperature. After rinsing, sections were dehydrated through a graded ethanol series (35%, 50%, 70%, 80%, 90% for 5 min each, 95% for 10 min, and 100% for 30 min). The sections were then infiltrated via incubations with increasing concentrations of PolyBed812 in propylene oxide. After infiltration, sections were embedded in 100% PolyBed812 overnight at 60 °C in a drying oven. 100 nm ultrathin sections were cut using a Leica EM UC6 ultramicrotome and captured on Pioloform carbon-coated 1 × 2 Cu slot grids (EMS) and contrasted with Reynolds lead citrate and uranyl acetate. The sections were examined on a Phillips CM120 transmission electron microscope and images captured with a MegaView III digital camera (Olympus-SIS).
Statistical analysis
Specific methods for statistical analysis have been indicated in the Fig. captions with the number of replicates analyzed in each condition. A combination of one-way ANOVA paired with Tukey’s post-hoc analysis and two-tailed students t test were used based on the experiment. Statistics were calculated using excel and GraphPad Prism.
