Envision anti rabbit hrp

The patients fasted overnight prior to undergoing a gastroscopy. During the gastroscopy, four tissue biopsies were collected from the descending part of the duodenum, distal to the papilla. The biopsy samples were fixed in formaldehyde and embedded in paraffin. The biopsies were cut into 3 μm thick sections. Antigen retrieval was performed in the PT-Link® system for 20 minutes at 98°C with the Dako® EnVision FLEX Target retrieval buffer at pH 6 (K8005; Dako, Jena, Germany). Peroxidase blocking solution was used for 10 minutes. Thereafter, further blocking with 5% goat serum (X0907; Dako) in 3% bovine serum albumin for 30 minutes was performed. A one-hour incubation with a 1 : 50 dilution of the primary polyclonal rabbit antibody to guanylate cyclase type B was used (GUCA2B, no. LS-C371347, LifeSpan BioSciences Inc., Seattle, WA, USA). The secondary EnVision HRP anti-rabbit (K4011; Dako) antibody was incubated for 30 minutes. The slides were incubated with 3,3′-diaminobenzidine (DAB) solution for 8 minutes followed by counterstaining with hematoxylin.

Xenograft tumours of NOG mice engrafted with PLR327F-LD41 and MCC001F cells were fixed with 4% paraformaldehyde (PFA) on ice for 24 h and embedded in paraffin using the AMeX method^{28 (link)}. Thin sections were prepared for histopathology and immunohistochemistry. Histopathologically, HE-stained slides were prepared and read under a light microscope. For immunohistochemical staining of CK5, an anti-human CK5 antibody (clone EP1601Y, Abcam) was applied as the primary antibody after antigen retrieval in Target Retrieval Solution (Dako), followed by a labeled polymer reagent (Envision + HRP anti-rabbit, Dako) as the secondary antibody, and visualised by DAB solution. The slides were counterstained with hematoxylin and read under a light microscope (Eclipse Ni-U microscope equipped with DS-Ri2 digital camera, Nikon).
For the evaluation of 3D cultures, paraffin blocks of Mix-gel-cultured COs were prepared using a method described previously^{29 (link)}. Briefly, the culture was treated with 0.1 mg/mL Brightase-C for 30 min at 37 °C, mechanically disrupted, fixed with 4% PFA for 2 h at 4 °C, and the gel was further dissociated by pipetting. Then the COs were embedded in paraffin by the AMeX method. HE slides and immunohistochemical staining for CK5 was carried out with the same procedure as the xenograft tumours.

Slices were incubated 2 x 5 min in xylene (ThermoFisher, X/0200/21), 2 x 3 min in isopropanol (VWR, 20842.330) and 10 min in 1% H₂O₂ (VWR, 23.613.446) / methanol prepared extemporaneously. Slices were washed 3 min in tap water and 3 min in demineralized water. Slices were then incubated 30 min in the 98°C water bath with the “Target Retrieval Solution 1X” pH 6.1 (Dako, S169984-2) and let to cool down for 15 min at room temperature. Slices were washed 5 min in tap water, 2 x 3 min in PBS and incubated 1 h at room temperature with a solution of PBS-2% Normal Goat Serum (NGS) (ThermoFisher, 16210064). Slices were then incubated with the primary antibody anti-MPV17 (Table 2) diluted 1/100 in a solution of PBS-0.5% NGS, overnight at 4°C. Slices were washed 3 x 3 min in PBS-T 0.05% baths and 3 min in PBS. Slices were then incubated 30 min at room temperature with the secondary antibody EnVision-HRP anti-rabbit (Dako, K400311). Slices were washed 3 x 3 min in PBS-T 0.05% and 3 min in PBS. Slices were then incubated 4 min at room temperature in the DAB solution (Dako, K346811) and washed 5 min in running tap water. Slices were incubated 5 min in Mayer Hematoxyline and washed 5 min in running tap water. Slices were finally incubated 3x 3 min in isopropanol, 3x 3 min in xylene and mounted on coverslip with Entallan glue.

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.