Vectamount

For immunohistochemistry analysis, frozen OCT-embedded liver biopsies from HDV-infected patients and controls were sectioned into 5-μm tissue sections, placed on SuperFrost Ultra Plus slides (Histolab) and stored at –80°C until staining. The sections were fixed with 4% paraformaldehyde (Sigma Aldrich) for 20 min on ice, followed by two blocking steps: one for elimination of endogenous peroxidase activity, using Bloxall (Vector Laboratories), and another one for reduction of general background staining, using Innovex background buster (Innovex Biosciences). Blocking was performed at room temperature (RT), for 10 and 20 min respectively. Thereafter, sections were incubated with purified rabbit anti-human CD3 epsilon (EP449E, Epitomics) or mouse anti-human TCR Vα7.2 (3C10, Biolegend), with isotype-matched antibodies serving as the corresponding negative controls. Subsequently, sections were incubated with ImmPRESS mouse or rabbit reagent (Vector Laboratories) for 30 min at RT, followed by signal detection using ImmPACT DAB as the peroxidase substrate solution (Vector Laboratories). Tissue sections were counterstained with Hematoxylin (Histolab) and mounted with Vectamount (Vector Laboratories). Immunoreactivity was visualized and analyzed by light microscopy (Leica DM 4000B).

Mouse brains were sectioned sagittally down the midline and fixed in formalin. Tissue blocks were paraffin-embedded and cut into 5µm sections. Following antigen retrieval, tissues were blocked and incubated with α-Ki67 (1:500; Vector, VP-RMO4) or Ccnd1 (1:100; Thermo Scientific, SP4) overnight at 4°C, then incubated with biotinylated goat anti-rabbit IgG (1:300; Vector, BA-1000). Slides were stained with hematoxylin and mounted using VectaMount (Vector). Images were captured with an Olympus DP70 digital camera, and were analyzed using the DP Controller software package (Olympus). Images were processed for publication using Adobe Photoshop Elements 5.0 (Adobe Systems).

Amygdala, hippocampal, and SMTG specimens were fixed in 10% formaldehyde, embedded in paraffin, and cut into ~8 μm thick sections. Slides were baked in a 40°C oven overnight, deparaffinized using SafeClear (Fisher Scientific), and rehydrated through a series of graded alcohols to water. Slides were boiled for 10 minutes in Borg Decloaker antigen retrieval solution, pH = 6.0 (Biocare Medical) before blocking endogenous peroxidase activity with 3% hydrogen peroxide + 10% methanol. The slides were blocked (0.1 M Tris buffer with 0.1% Triton X-100 and 2% bovine serum albumin) and incubated overnight at 4°C in the foll owing primary antibodies: 1:25 rabbit polyclonal anti-ΔCN; 1:500 mouse monoclonal anti-CN-Aα (C-terminus) (Sigma);. 1:50 mouse monoclonal anti-Aβ (Vector Laboratories. Secondary antibodies were added at a 1:200 dilution for 1 hour at room temperature as follows: anti-mouse IgG + horse normal serum or anti-rabbit IgG + goat normal serum. Following secondary antibody, signal was amplified with avidin-biotin complex for 1 hour at room temperature and then detected using either DAB or SG substrates (Vector Laboratories). Following dehydration through a series of graded alcohol and clearing (SafeClear), slides were permanently mounted using VectaMount (Vector Laboratories) and coverslipped.