3 3 diaminobenzidine chromogen solution

Brain tissues were fixed in AntigenFix solution (Diapath, France) for 24 h. Then, they were decontaminated for 30 min in formic acid solution according to the protocol described by Andréoletti et al. [37 ] and stored in 100 mM phosphate buffer at pH 7.4 with 0.02 % sodium azide. Samples were dehydrated in graded ethanol, cleared in cedar oil and embedded in paraffin. Frontal 6- to 10-μm sections were cut using a microtome and mounted on Superfrost Plus slides (Microm France, Francheville). Sections were dewaxed and stained with hematoxylin and eosin as described previously [38 (link)]. Immunolabeling with anti-GFAP antibodies was performed according to the instructions provided with the Strept ABC Complex Kit. Labeling was visualized using 3-3’-diaminobenzidine chromogen solution (Sigma, France). For paraffin-embedded tissue blots (PET-blots), 6 μm frontal sections were cut using a microtome and placed on nitrocellulose membrane. After drying at 50 °C for 48 h, sections were dewaxed, digested with 25 μg/mL PK at 56 °C overnight and then denatured with 3 M guanidine thiocyanate for 10 min. Membranes were blocked with casein for 30 min. The SAF84 antibody was used to label rPrP^Sc and the Vectastain ABC-AmP kit (Vector laboratories, USA) to reveal antibody binding.

Brain tissues were fixed in AntigenFix solution (Diapath, France) for 24 h. Then, they were decontaminated for 30 min in formic acid solution according to the protocol described by Andréoletti et al.⁴⁵ and stored in 100 mM phosphate buffer at pH 7.4 with 0.02% sodium azide. Samples were dehydrated in graded ethanol, cleared in cedar oil and embedded in paraffin. Using a microtome, 6 μm frontal sections were cut and mounted on Superfrost Plus slides (Microm France, Francheville). Sections were dewaxed and stained with hematoxylin and eosin (HE) as described previously^{46 (link)}. Immunolabelling with anti-GFAP (1:500; Dako, Les Ulis, France) antibodies was performed according to the instructions provided with the Strept ABC Complex Kit. Labelling was visualized using 3–3′-diaminobenzidine chromogen solution (Sigma, France). For paraffin-embedded tissue blots (PET-blots), 6 μm frontal sections were cut using a microtome and placed on nitrocellulose membrane. After drying at 50 °C for 48 h, sections were dewaxed, digested with 25 µg/mL PK at 56 °C overnight and then denatured with 3 M guanidine thiocyanate for 10 min. Membranes were blocked with casein for 30 min. The SAF84 antibody was used to label PrP^Sc and the Vectastain ABC-AmP kit (Vector laboratories, USA) to reveal antibody binding.

Deparaffinized kidney sections (4 μm) were blocked for endogenous peroxidases. Sections were stained with either rabbit anti-mouse (Biodesign, Saco, ME) or rabbit anti-mouse collage IV. After 1 h, the slides were washed and incubated for 30 min at room temperature with biotinylated-labeled goat anti-rabbit, followed by Vectastain ABC reagent (Vector Labs, Burlingame, CA) and 3,3'-diamino-benzidine chromogen solution (Sigma, St. Louis, MO). The sections were examined and graded on a scale of 0 to 4, as previously described [26 ], by a renal pathologist (GS) who was blinded to the treatment group.