Vector blue

Calcified sections were prepared and stained according to the previous report [75 ]. Briefly, specimens attaching to the cryotape were glued on the glass slides with chitosan adhesive solution, and dried overnight at 4 °C. As for TNAP staining, slides were incubated for 10 min in 100 mM Tris-HCl buffer (AP Buffer) (pH 8–8.5) mixed with Vector Blue (Vector laboratories, SK-5300) at room temperature following the instruction of the manufacturer. Subsequently, the slides were rinsed in 1X PBS for 5 min three times and mounted with DAPI and visualized under the Cy5 channel. For TRAP staining, ELF97 (Thermo Fisher Scientific, E6588) was diluted 1:75 in TRAP buffer (9.2 g of sodium acetate anhydrous and 11.4 g of sodium tartrate dibasic dihydrate dissolved in water; total volume 1000 ml; pH 4.2). TRAP buffer was applied to the slides for 15 min at room temperature. Subsequently, ELF97 dissolved TRAP buffer was applied to the slides for minutes at room temperature. The slides were rinsed in 1X PBS for 5 min three times and mounted with DAPI and visualized under the GFP channel.

Nonspecific binding was blocked with Blocking One (Nacalai Tesque) for 30 min at room temperature. A sequential double staining procedure^{50 (link)} was initiated with CitFbg, and was visualized by BrightVision HRP-conjugated anti-mouse IgG polymer (DPVM-HRP, Immunologic) and Bright DAB (BS04, ImmunoLogic). The DAB reaction product effectively sheltered the first set of antibodies. Then, the process was continued with VE-cadherin (1:200, sc6458, Santa Cruz) and anti-human SAA1/2 (1:1000, ab207445, Abcam) and anti-human Fbg (1:1000, A0800, Dako), and was visualized by BrightVision alkaline phosphatase (AP-)conjugated anti-rabbit IgG polymer (DPVR-AP, ImmunoLogic) and Vector Blue (SK-5300, Vector Laboratories; Burlingame, CA). The slides were counterstained, washed with tap water, and coverslipped with PathodMount (Fujifilm Wako) after dehydration with Clear-Rite 3^TM (Richard-Allen Scientific LLC, USA). Images were acquired with Vectra 3 (PerkinElmer), Phenochart 1.1.0, and inForm 2.4.9 (Akoya Bioscience).

Membrane plates (0.45 μm Hydrophobic High Protein Binding Immobilon-P Membrane, MilliporeSigma) were coated with 5 mg/mL of heat-killed inactivated Nme and placed at 4°C overnight. The plates were blocked the next day with 10% FBS/complete RPMI for at least 2 hours at 37°C. Single-cell suspensions were loaded onto the plate at serial 2-fold dilutions in 10% FBS/complete RPMI and incubated overnight at 37°C. Cells were removed the next day and washed with 0.1% Tween 20/PBS 5×. HRP-conjugated IgA and AP-conjugated IgG (in the case of 2-color ELISPOT) detection antibodies were subsequently added for 2 hours at 37°C.
Plates were then washed with 0.1% Tween 20/PBS 3× and with PBS 3×. The plates were developed while covered with aluminum foil until spots were visible using 3-amino-9-ethylcarbazole (AEC) peroxidase (for HRP-conjugated antibodies, Vector Laboratories) and Vector blue (for AP-conjugated antibodies, Vector Laboratories) substrates. After the development was done, the plates were washed with distilled water and were left to dry overnight. The spots were counted based on the original cell dilution.

We used paraffin embedded frontal lobe brain sections from three deceased CADASIL patients (I: female age 59, p.Arg153Cys; II: female age 57, p.Arg153Cys; III: male age 70, p.Cys446Phe) and three deceased controls with no known cerebrovascular disorders (I: male age 67, II: male age 58, III: male age 53). Sections were de-waxed, rinsed with ethanol and blocked with methanol/H₂O₂. After heat-induced antigen retrieval in 0.01 M citrate buffer pH 6, slices were washed three times with PBS, and incubated overnight at room temperature with a 1:1 cocktail of anti-NOTCH3^ECD (dilution 1:500) and anti-CD31 (Dako, Glostrup, Denmark; dilution 1:50). The following day, sections were washed and incubated for 1 hour at room temperature with a 1:1 cocktail of anti-rabbit Envision/HRP (Dako) and goat anti-mouse alkaline phosphatase (Vector Laboratories, Burlingame, CA, USA; dilution 1:25). Finally, sections were sequentially developed with 3,3′-diaminobenzidine solution and Vector Blue (Vector laboratories). Per individual, four images were taken at a 400× magnification on a Leica IM 500 microscope and analysed using ImageJ software. The vessel area was selected manually based on a positive CD31 staining. Within the vessel area, the NOTCH3 score was calculated using an intensity threshold of 100.

The co-localization of the a DC marker with a marker for cross-presentation in human plaques was measured by multispectral imaging of immunohistochemical staining. Frozen human plaque sections were stained for CD11c (BD Pharmingen) and XCR1 (Novus Biologicals). From double staining, spectral imaging data sets from maximal three random regions of interest were taken between 420–720 nm (10 nm interval) at a 5× as well as at a 20× magnification using a Nuance spectral imaging system (Perkin Elmer/Caliper Life Sciences, Hopkinton, MA, USA) mounted on a Zeiss Axiophot microscope. Slides stained for a single chromogen (Vector Red and Vector Blue, both Vector Laboratories) only were used to create a spectral library. The spectral library was used for computational segregation of the individual image components using the NuanceTM 3.0.2 software as described59 (link). After spectral unmixing, pseudo-colors were assigned to unmixed images, and composite images showing co-localization were generated with the Nuance 3.0.2 software.