Treatment of pellucida of embryos by TACIT and the RNA-seq method for control of tertipotency-related transcription factors

The zona pellucida of embryos were removed by treating them with pre-warmed Tyrode’s acidic solution. The embryos were fixed with M2 medium after being transferred to it. The embryos were stored at −20 °C or immediately used. The regular TACIT was directly applied to the whole embryos. Two cells from the same embryo were separated and deposited into different wells of lysis buffer after tagmentation. Every well was covered with 5 l mineral oil and placed in a body of water for 15 minutes. Next, 0.2 l of 10 mM. Adding PMSF and 1 l of Triton X-100 in each well made a difference to the protease K reaction. PCR amplification was conducted as described for TACIT libraries, and DNA fragments of the two cells from the same 2cell embryos were barcoded with different combinations of Nextera i5 and i7 indexes. Finally, the embryo-barcoded TACIT library was sequenced as described for conventional TACIT.

The sgRNAs targeting the promoter of each of the candidate totipotency-relatedTFPs were inserted into a CROP-optivector and synthesised. Three libraries of sgRNAs included candidate TFs (CEBPG, LBX1, ETS2, MEF2D, ESR2, ESR1 and ALX1), positive control TFs (ZSCAN4 and DUX) and a non-targeting control as previously described53 at equal molar ratios. The supernatant with lentivirus was collected 18 h after transfection and filtered to remove cell debris. The mouse ES cells were infected (8 μg ml–1 polybrene) with various titres of lentivirus to achieve different multiplicity of infection values. After 24 h, new culture medium with 2 g ml–1 puromycin was added. Cells were collected for scRNA-seq after they were transduction and selection. The cells were fixed with 1% of formaldehyde for 10 minutes and then preserved at 80 C. Single-cell RNA-seq for mouse ES cells for capturing both mRNA and sgRNAs was conducted as per the SPLiT-seq pipeline as previously described54.

The expected probability was used in the calculation of the enrichment of the bins. The expected probability was calculated using the length of the total bins against the length of the related genomic regions. The promoter was a region around all the TSSs. The locations of annotated repeats (RepeatMasker) were downloaded from the UCSC Genome browser18,64.

Source: Genome-coverage single-cell histone modifications for embryo lineage tracing

Random Forest Model 62: Interpolating Heterogeneous Single Cells in the Late 2 Cell Stage using the HiC Function of Homer and python

The late 2cell stage allvalid pairs matrix was downloaded from the GEO database. The analyzeHiC function of Homer was utilized to identify interactions and the python program was used for plotted interactions.

We integrated H3K4me1, H3K4me3, H3K36me3 and H3K27ac TACIT profiles with gene expression. The histone modification matrices were first generated using cisTopic58. The GeneActivity function of Seurat (v.4) was used to create a gene-activity score matrix based on the cell–peak or cell–bin matrix. Next, anchors between the two modalities were identified with the FindTransferAnchors function. In particular, many titrations were performed to obtain the highest prediction score, including using the cell–peak or cell–bin matrix, or the bin size of the cell–bin matrix. TACIT cells with a prediction score lower than 0.2 were filtered out. Histone-modification signals in non-Canonical broad binding regions were not included in the integration of cells in the 2cell stage.

Two strategies were used to rule out differences in stage between the 2cell 1 and 2cell 2 bins. Each strategy had a cell-bin probability matrix created for each state, and a correlation of at least 50% with the expression of totipotency marker genes selected. Next, the highly correlated candidate bins were aggregated to create the state matrix. The state matrix of synthetic cells at the 2cell and 8cell stages was used as input for constructing the random forest training model62, with labels as ‘toti-high’ and ‘toti-no’ groups, respectively.

To evaluate cell heterogeneity among stages for each histone modification, we first calculated the Euclidean distance between each pair of cells in the same stage as shown in the UMAP embeddings. The median Euclidean distance of zygotes was set as the baseline for normalization of other cells across all stages.

We used the LearnModel command of ChromHMM46 to train a 12-state model with aggregate ICM and TE profiles. Next, we ran the forward–backward algorithm to learn the posterior probability distribution for interpolated single cells. The states were grouped into categories by the bin size and they were H3K9me3 associated heterochromatin and quiescent/low. We also merged five adjacent single cells along pseudotime52.

Having obtained 155 RNA synthetic cells interpolated with six histone-modification profiles, we performed hierarchical clustering with RNA synthetic cells on the basis of multimodal histone modifications. The number of clusters closely corresponded to the exact cell number of each developmental stage, such as two clusters for the 2cell stage, four clusters for the 4cell stage, and so on. 90 synthetic single cells with joint histone modifications were created as a result of we aggregated histone-modification profiles of cells in the same cluster. We normalized cell numbers and non-duplicated reads to make it easier to understand the data.

We did not make any changes in the way H3K 27ac CoTACIT profiles are integrated. As H3K27ac, H3K27me3 and H3K9me3 profiles were experimentally linked, we directly transferred corresponding H3K27me3 and H3K9me3 profiles to the linked RNA synthetic cells.

We merged five cells along a pseudo time path into oneRNA synthetic cell, using Monocle3 to do it.

To calculate the genome coverage at each developmental stage, we first called peaks for aggregated .bam files of each histone modification. We used MACS2 to call peaks with parameters of ‘–nolambda–nomodel -q 0.05–broad’. The percentage of genome intervals that coincide with peaks at each stage of the histone modification was calculated by taking into account the binned genome’s 200bp intervals. To evaluate the coverage of the genome, it was first binned into 200 bp and bins with histone modification signals defined as covered bins. The percentage of covered bins was defined as genome coverage for each single cell.

For correlation analysis between different experiments, we calculated the normalized mean scores in 5-kb bins of the genome by using the multiBigwigSummary function in deepTools (v.3.5.1)57. The Spearman correlation or Pearson correlation was calculated between replicates and plotted using the plotCorrelation function.

Preparation and Treatment of Isolated CoTACIT with Embryos for Developmental Stage 1 CoTIT with Single Cells (Diagram Report)

For CoTACIT with embryos, isolated single cells were rehydrated and washed as described above. The cells were put into a 100 l buffer with H3K4me3 or H3K29ac for the early 2cell stage or H3K29ac for all six developmental stages. NaCl, spermidine, and 0.1 mM. 3 h at 4 C for 0.01% digitonin, 0.05% TX-100, 1%PBS, 10 mMsodium butyrate and 1 mM PMSF. The cells were washed again with 180 l. Dig-wash buffer (20 mM HEPES pH 7.5, 150 mM 1 cocktail, 10 mM sodium butyrate and 1 mM PMSF are included. Cells were given a high-salt Dig-wash buffer and 3 liters of cells were put to use. NaCl, 0.5 mM spermidine, 0.01% digitonin, 0.05% TX-100, 1× cocktail, 10 mM sodium butyrate and 1 mM PMSF) at 4 °C for 1 h and washed twice with 180 μl high-salt Dig-wash buffer. After tagmentation and inactivation with 20 mM Three times, cells were washed with a small amount ofPBS to remove free PAT and adapters. The second round of barcoding was performed as for the first round, except that cells were incubated with 0.5 μg H3K27ac (for the early 2cell stage) or 0.5 μg H3K27me3 (for all of six developmental stages) in 100 μl antibody buffer and barcoded with 3 μg ml–1 PAT-T5-2 and 3 μg ml–1 PAT-T7-2 in 100 μl high-salt Dig-wash buffer at 4 °C for 1 h. The same procedure was used for the third round of barcoding, except that the cells were put into a pot with a higher concentration of H3K9me3 than in the previous round. A 96-well plate was prepared in which single cells were picked, fragment release and the quenching of the SDS were carried out after being washed 3 times.

The 1 l cDNA was used to measure the concentration. About 10 ng was tagged with a single MEA in a buffer of 10 mM, and also with 10 mM and 10% N,N-dimethylformamide. Samples were then treated with 0.025% SDS at 55 °C for 10 min and 0.15% Triton X-100 at 37 °C for 10 min. 3 min at 95 C was used for initial denaturation, followed by 16 cycles of 20 s at 98 C and 1 min at 72 C, and 5 min at 72 C. The library was cleansed with beads from AMPure. The size selection was done first with the 0.5 AM Pure beads in the Supernatant, and then with the second with the 1 AM Pure beads in the supernatant. The libraries were read with 150-bps reads on the NovaSeq 6000 platform.

For siRNA knockdown, isolated zygotes were microinjected with sets of three siRNAs against targets (20 μM in total) or with non-target control (NC, 20 μM in total). The following siRNAs were used. Hippobio ordered the siRNAs. The injected embryo was transferred to KSOMaa medium, where it was covered with mineral oil, and cultured in a tissue incubator that was 37 C and 5% CO2. Embryos were collected at the 8cell or blastocyst stage, and single-embryo RNA-seq or immunofluorescence staining was performed to confirm KD or marker gene expression.

The following antibodies were used for TACIT (catalogue and lot numbers provided after the supplier name): H3K4me1 (1:50; Abcam, ab8895, GR3369516-1); H3K4me3 (1:200; Millipore, 04-745, 3243412); H3K27ac (1:500; Diagenode, C15410196, A1723-0041D); H3K36me3 (1:200; Active Motif, 61101, 06221007); H3K27me3 (1:200; Millipore, 07-449, 3146226); H3K9me3 (1:200; Active Motif, 39161, 30220003); and H2A.Z (1:200, Abcam, ab4174, GR279096-1). Donkey anti-rabbit-Alexa 488 (1:500; Invitrogen, A32790) and donkey anti-rabbit-Alexa 555 (1:500, Invitrogen, A31572) were used as secondary antibodies. The SOX2 and CDX2 were Antibodies used in immunofluorescence staining.

The cells had been cultured at 37 C with 5% CO2 and were put on high-glucose plates that contained 15% Fetal bovine serum and 1% penicillin.

Source: Genome-coverage single-cell histone modifications for embryo lineage tracing

Experimental protocols for the conduct of animal experiments in Peking University on a 12 hr light-dark cycle: data handling and statistical analyses

To collect the zygotes, a large mass of them was transferred to a 1 hyaluronidase solution and incubated at 37 C for a few minutes. The zona pellucida was gently removed after the sperm was transferred to M2 medium. The second polar bodies of zygotes were manually removed with a very fine glass needle.

The protocols for conducting animal experiments were approved by the Institutional Animal Care and Use Committee of Peking University. All mice were maintained in pathogen-free conditions at the Laboratory Animal Center of Peking University on a 12–12-h light–dark cycle, with a temperature of 20–25 °C and humidity of 30–70% and access to food and water ad libitum.

Data handling and statistical analyses were done in a way that used R studio and GraphPad Prism Software. The analyses were done using the un-tailed two-tailed Student’s t-tests. All analyses were carried out using one-way ANOVA tests with post hoc tests. A P value of <0.05 was considered significant. In order to avoid temporal and technical biases, all procedures involving multiple groups were carried out in alternating fashion. Data in Figs. 1b–d,h–o, 2a,b,f–j, 3e–n, 4f–i and 5j–l and Extended Data Figs. 2a, 3f–i, 7a–c,h,i and 8h–k were successfully replicated in at least two independent experiments.

In fear conditioning tests, mice were trained to associate cage context and an audiovisual cue with an aversive stimulus (foot shock). On the first day of training mice were put in a cage and exposed to two periods of 30 s of cue light and 1,000-hertz tone and then a foot shock with a 180-s interval. On day 2 (contextual fear conditioning), mice were re-exposed to the same cage context and freezing behaviour was recorded during minutes 1.5 to 6 using a FreezeScan tracking system (Cleversys). On day four mice were placed in a novel context with different odor, floor texture, and chamber walls and re-exposed the same cue light and tone from day one after 2 minutes of exploration. The freeze behavior was recorded for 6 minutes using the FreezeScan tracking system.

In the Y maze test, the Y maze is made up of 3 white, opaque plastic arms at 120° angles from each other. Mouse were placed in the end of one arm, and then allowed to explore all 3 arms for 5 minutes. An arm entry was defined as having all four limbs inside an arm. The maze was cleaned with 70% ethanol between animals and before the first animal to eliminate traces of odour. The number of arm entries and triads (a set of consecutive arm entries) were recorded. The number of combinations and the number of possible combinations were used to calculate theSpontaneous Alteration.

Determination of pseudobulk counts and differential gene expression based on the Benjamini-Hochberg procedure for M. musculus

Pseudobulk counts were derived by aggregating raw counts for each sample using Seurat’s AggregateExpression function. Bulk data normalization and differential gene expression were performed using default parameters. P value correction was carried out using the Benjamini–Hochberg procedure (FDR = 0.05) for each comparison. Genes with FDR were used for the enrichment of the KEGG pathway.

Gene counts were obtained by aligning reads to the M. musculus reference genome GRCm38 using CellRanger software (v4.0.0) (10X Genomics). The nuclei were removed from each sample using SoupX and DoubletFinder. We used Seurat to exclude cells with less than 200 features and cells with more than 10% of their genes. In total, 69,250 nuclei remained and were used for further analysis (Supplementary Fig. 3a–d).

Source: Glycocalyx dysregulation impairs blood–brain barrier in ageing and disease

Dissociation and mechanical cleavage of microvessels from young mouse brains in PBS with 1c Oxplete protease inhibitor cocktail (Millipore Sigma) on ice

Microvessels from young (3-month-old) and aged (21-month-old) mice were isolated as previously described with some modifications26,34. In brief, mice were euthanized via CO2, and brains were retrieved in PBS supplemented with 1% bovine serum albumin (BSA) and 1× cOmplete protease inhibitor cocktail (Millipore Sigma) on ice. The meningeal vessels were removed by rolling on blotting paper. Brains were minced using a razor blade on ice and then homogenized using a loose-fit, 7 ml ShakeWheaton with the 1 protease blocker 1% BSA-PBS. The homogenate was centrifuged in 27% (wt/vol) 70 kDa dextran (Sigma) in HBSS at 4,400g for 25 min. Myelin and parenchymal cell layers were removed. Microvessels were washed with PBS and mechanically cleaved into single cells on a 40-m strainer. Previously published protocols were used for the dissociation of brain tissue. Pelleted cells were suspended in FACS buffer (1% BSA in PBS) and stained on ice for 30 min with the following antibodies: rat anti-CD31-PE/CF594 (1:100, BD, 563616), rat anti-CD45-PE/Cy7 (1:200, Biolegend, 103114), mouse anti-heparan sulfate (1:100, Amsbio, clone 10E4, 370255-1), mouse anti-chondroitin sulfate (1:100, Sigma, clone CD-56, C8035), biotinylated HABP (1:150, Amsbio, AMS.HKD-BC41), fluorescein-conjugated SNA (1:300, Vector Labs, FL-1301-2), biotinylated MAAII (1:300, Vector Labs, B-1265-1), and StcE(E447D)–AF647 (5 μg ml−1). Secondaryincubation with conjugated penicillin (1:1,000) and secondarylizumab (1:400) was carried out on ice for 20 min after washing cells. Live cells were identified with the help of a viability dye. Data was analysed using FlowJo software and the analysis of flow cytometry was performed on a LsrForta.

The Addgene plasmid 127807 was given to V. Gradinaru as a gift for the production of thePHP.V1. The transfection of HEK293T cells was done on 90-93 percent confluent cells with 4% FBS and non-essential Amino acids in the form of Glutamax. After thefection, the warm medium was replaced. The medium was collected 72 hours after thefection. Fresh, warm medium was added and collected along with cells 120 h post-transfection and combined with the previous fraction. Medium and cells were placed at a temperature of 2,000g for 15 minutes. 40% was combined with a separate bottle of Supernatant. After 2 hours on ice, it was time to transfer the final concentration to 4 C. A buffer containing a salt- active nuclease was used to keep the cell pellet in for a while before it was transferred to 4 C. PEG medium was centrifuged at 4,000g for 30 min at 4 °C. After centrifugation, supernatant was bleached and discarded. The PEG pellet was resuspended in the SAN + SAN buffer and incubated at a higher temperature of 37 C. Lysate was centrifuged at 2,000g for 15 min at room temperature, and supernatant was loaded onto an iodixanol gradient (15%, 25%, 40% and 60% fractions). Gradients were transferred to an ultracentrifuge (Beckman Coulter) using a Type 70 Ti rotor set at 350,000g for 2 h and 25 min at 18 °C. AAD particles were washed in PBS and concentrated using the Amicon Ultra-15 filter device, which has a 100 kDa cutoff. AAV titration was performed using the AAVpro Titration Kit (for Real Time PCR) Ver.2 (Takara Bio). AAVs were injected retro-orbitally at 8 × 1011 viral genomes per mouse.

The Ple261 MiniPromoter is a gift from E. Simpson. A cis rAAV genome plasmid with AAV2 inverted terminal repeats was utilized for cloning of a sCLDN5 and EGFP reporter using restriction enzymes and In-Fusion Snap Assembly (Takara Bio). To knock down C1galt1 in brain endothelial cells, de novo predictions of small interfering RNA (siRNA) guides targeting C1galt1 were generated using the DSIR algorithm39 and subsequently filtered using ‘Sensor rules’ to select for sequences with highly favourable small hairpin RNA (shRNA) features40,41. Three de novo 97-mer miR-E shRNA sequences were created and inserted into pAAV-sclDN5-EGFP for evaluation. To overexpress C1GALT1 and B3GNT3 in brain endothelial cells, P2A-C1GALT1 and P2A-B3GNT3 were cloned into pAAV-sCLDN5-EGFP using restriction enzyme cloning to generate pAAV-sCLDN5-EGFP-P2A-C1GALT1 and pAAV-sCLDN5-EGFP-P2A-B3GNT3, respectively.

Cerebral microvessels were isolated for immunofluorescence imaging using the same protocol described above for flow cytometry, except, instead of dissociating microvessels, they were fixed on 40-μm strainers with 4% PFA in PBS at room temperature for 15 min with gentle rocking. Microvessels were then washed with PBS and mounted on poly-d-lysine-coated slides (Thermo Fisher Scientific). Microvessels were blocked in 3% normal donkey serum (Jackson ImmunoResearch) with 0.3% Triton X-100 (Sigma) in TBS-T (1× TBS with 0.05% Tween-20) for 1 h, followed by 1 h incubation at room temperature with primary antibodies. The same primary antibodies, binding proteins and concentrations from the flow panel were used along with the fluORESCEin-conjugated VVA for the purpose of circulating cerebral microvessels. Rabbit anti-C1GALT1 (1: 100) is one of the additional primary antibodies used. Microvessels were washed three times with a TBS-T for 5 min each, and then taken to the lab for 1 h at room temperature or a Fluor conjugated antibodies for 1, then taken to the lab for 1 h. Microvessels were washed three times and then covered with a Vecta shield Hardset Antifade mounting medium. Imaging was performed on a confocal laser-scanning microscope (Zeiss LSM880). All observed single-plane microvessels were quantified using ImageJ software.

For confocal imaging analysis, bEnd.3 cells were plated on round coverslips (EMS, 72196-12) in a 24-well plate and treated with 5 nM StcE for 16 h at 37 °C. Cells were fixed in 4% PFA for 15 min, blocked in 3% normal donkey serum with 0.3% Triton X-100 in PBS for 1 h at room temperature, and incubated at room temperature in blocking solution with the following primary antibodies for 1.5 h: goat anti-CD31 (1:100, R&D, AF3628), mouse anti-ZO1 (1:100, Thermo Fisher Scientific, 33–9100), rabbit anti-CAV1 (1:100, Cell Signaling Technologies; 3267S) and mouse anti-CLTC (1:100, Thermo Fisher Scientific, MA1-065). Cells were subsequently washed 3 times with PBS, stained with the appropriate Alexa Fluor-conjugated secondary antibodies (1:250, Thermo Fisher Scientific) for 1 h at room temperature, washed 3 times again, mounted, and coverslipped with Vectashield Hardset Antifade Mounting Medium with DAPI (Vector Labs, H-1500-10) or ProLong Gold Antifade Mountant (Thermo Fisher Scientific, P36934). A confocal laser-scanning microscope was used to perform and analysis of images.

The mouse brain endothelial cell line bEnd.3 was kept in a humidified incubator, where it was cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin. BEnd.3 cells were grown in 6well plates and treated with 5 nM StcE for 16 h at 37 C. The cells were collected and lysed using the Eppendorf tubes and the Qiagen kit. The quantity and quality of the RNA was assessed by a Bioanalyzer. All samples passed a high quality control threshold (RNA integrity number ≥9.7) and proceeded to cDNA library preparation by Novogene. Libraries were sequenced on the NovaSeq 6000 (paired-end, 2× 150 bp depth). The reads were aligned to the reference genome. Differential gene expression analysis and visualization were performed using DESeq2 (v1.32) (Supplementary Data 5). GObiological pathway enrichment analysis was done using genes with a Padj 0.05.

Previously published ageing and neurodegenerative disease RNA-seq datasets demonstrating robust brain endothelial cell enrichment were chosen for glycosylation-related gene analysis12,26,27. We filtered for glycosylation-related genes based on KEGG (Kyoto Encyclopedia of Genes and Genomes) listed glycosylation enzymes and related proteins. The huge variety of members in this family have biological functions not relevant to the study of glycosylation were the reason most glycoproteins were excluded. The threshold for significant enrichment was set by Padj at 0.05 in each dataset.

The LC–MS/MS analysis was performed on a Q ExactiveHF-X forThermo Fisher Scientific. Peptides were loaded on an in-house 75-μm (inner diameter) capillary column packed with 40 cm of ReproSil-Pur 120 C18-AQ 1.9 μm resin (Dr. Maisch). The separation using a flow rate of 300 nl min1 took the following amount of time: 18 min, 70 min, 60%, 40%, and 4% A + 4% B. Full MS scans were acquired at a resolution of 60,000, with an automatic gain control (AGC) target of 3 × 106, maximum injection time (IT) of 20 ms, and scan range 300–1,650 m/z in a data-dependent mode. The resolution was 15,000, the AGC target was 1 105, the maximum IT was 54 ms, the loop count was 15, and the isolation window was 1.4 m/z. The Mus musculus reference proteome database was downloaded by Uniprot. Methionine oxidation and N-terminal acetylation were specified as variable modifications, and carbamidomethylation of cysteines was specified as a fixed modification. Precursor ion search tolerance of 20 ppm and product ion mass tolerance of 20 ppm were used for searches. Both unique and razor peptides were used for quantification. Results were filtered to a 1% false discovery rate (FDR) at the peptide and protein levels. The minimum ratio count for the MaxLFQ37 was set to 1. For the analysis, missing and log2 transformed values were imputed from a normal distribution with width 0.4 and downshift value of 1.7. Principal component analysis (PCA) was performed in Perseus using the Benjamini–Hochberg FDR with a cutoff of 0.05. GO term enrichments were performed using DAVID38 with the M. musculus proteome as a background.

Plasma cytokine measurement was performed using the Luminex assay at the Human Immune Monitoring Center at Stanford University. The mouse 48-plex Procarta kit (Thermo, EPX480-20834-901) was used according to the manufacturer’s instructions. Plasma samples were diluted 1:3 and run in singlet on a 96-well plate alongside standard curve and quality control samples. Custom Assay Chex control beads (Radix BioSolutions) were added to all wells to assess nonspecific binding.

StcE was produced as described. For all applications, there were at least seven times how many proteins could be run through a high-capacity endotoxin removal column. The endotoxin levels were tested using the InvivoGen kit. The mice were injected into their brains with 0.25 grams of StcE every day for 2 days before they were put to sleep. Cerebral bleeding was detected by eye post-perfusion and by H&E staining. For H&E staining, hemibrains and peripheral organs were formalin-fixed and paraffin embedded (FFPE) and cut into 5-μm-thick sagittal sections mounted on slides. Section were deparaffinized in xylene, then hydrated with a series of alcohols and stained with Richard Allan haematoxylin. Sections were dehydrated, they were cleared of xylene, and covered before being used on a wide-field microscope.

Mice were anaesthetized and injected retro-orbitally with Sulfo-NHS-biotin (Thermo Fisher Scientific, 21335) at 0.25 mg g−1 body weight. The tracer was allowed to circulate for 5 min before perfusion with PBS. The hiabrains were post-fixed with 4% PFC and then sectioned into 40-m slices. Sections were blocked and co-stained with CD31 and the appropriate secondary antibodies as described earlier. Images were taken on a confocal laser-scanning microscope (Zeiss LSM880) and analysed using ImageJ software. The permeability index of vessels was determined by dividing the area occupied by a tracer into the vessel area.

For luminal vascular labelling, mice were euthanized with 2.5% (v/v) Avertin and transcardially perfused via peristaltic pump at 2 ml min−1 with the following ice-cold solutions: 8 ml PBS, 10 ml of 5 μg ml−1 of StcE(E447D)–AF647 or SNA–Cy3 (Vector Labs, CL-1303-1), and 8 ml of 4% PFA. For all other immunofluorescence analysis, mice were euthanized with 2.5% (v/v) Avertin and manually perfused with PBS unless noted otherwise. Tissues were extracted and fixed in 4% PFA at 4 °C overnight before preservation in 30% sucrose in PBS. The 40 m slices of the tissue were divided by a microtome. After a 1.5 h at room temperature, the slices were blocked with 3% normal donkey serum with 0.3% Triton X-100 in TBS-T, and 4 C overnight with the following primary antibodies: goat anti-CD31 (1: 100, R&D, AF3628), goat anti The following day, slices were washed three times with TBS-T, stained with the appropriate Alexa Fluor-conjugated secondary antibodies (1:250, Thermo Fisher Scientific) or Alexa Fluor-conjugated streptavidin (1:1,000, Thermo Fisher Scientific) for 2 h at room temperature, washed three times again, mounted, and coverslipped with Vectashield Hardset Antifade Mounting Medium with DAPI (Vector Labs, H-1500-10). The confocal laser- scanning microscope was used to image and analyse the images. The marker of interest was used to divide the total vessel area by the vessel area for luminal vascular coverage. The CD31+ mask is used to calculate the endothelial MFI.

Post mortem fresh-frozen brain tissues were obtained from Stanford/VA Aging Clinical Research Center with approval from the Stanford Institutional Review Board and patient consent. The autopsy was done no later than 12 h after death and all samples were kept at 80 C until the time of processing. Group characteristics are summarized in Supplementary Data 2. Individuals in the Alzheimer’s disease group were both clinically diagnosed and pathologically determined to exhibit Alzheimer’s disease brain hallmarks including β-amyloid and tau pathophysiology.

The mice were injected with a small amount of HRP type II in PBS for the ultrastructural analysis. After 30 min, brains were fixed in a 0.1 M cacodylate buffer and for 1 h at room temperature. Tissues were washed overnight with 0.1 M sodium cacodylate at 4 °C and then sliced coronally using a matrix into 1-mm-thick sections. The Cortical punches were cut and put in a small container with a hydrogen peroxide concentration of less than1% and then put into a room for 45 minutes. After fixing in 2% osmium tetroxide and 2.5%potassium ferrocyanide in 0.1 M sodium cacodylate, the tissues were stained with 1% uranyl acetate and 10% Walton’s lead stain. Samples were then dehydrated in an ascending ethanol gradient and embedded in epoxy resin (EMS). The section was collected on formvar-coated copper grids and was cut using the UC7 ultramicrotome. The grids were washed with 3.5% uranyl acetate. Sections were imaged using a Tecnai 12 120 kv TEM, and the data was recorded using a Rio16 camera. The assessment of tight junctions in the images was performed in a blinded manner.