Aqueous mounting media

PLP fixed mouse brains were paraffin-embedded and 5 μm tissue sections were cut and mounted on glass positive charge slides. Tissues were deparaffinized in a 65°C oven followed by successive xylene immersions (100%), rehydrated through graded ethanol washes (100% x 2, 95% x 2 and 70% at 5 min per wash), and 88% formic acid treated for 60 min. Tissues were then subjected to hydrated autoclaving using an automated antigen-retrieval system 2100-Retriever (Prestige Medical) and a citrate buffer (0.01M sodium citrate, 0.05% tween 20, pH 6) for 30 min. Samples were then blocked with 3% H₂O₂ (30 min) followed by a proprietary protein block (TNB, PerkinElmer Life and Analytical Sciences) (30 min) and stained with unconjugated BAR-224 at 2 μg/ml (Cayman Chemical) (overnight at 4°C). Detection was completed using HRP-conjugated anti-mouse secondary antibody (Envision+, Dako) (30 min) and AEC substrate chromogen (Dako) (1 min). Tissues were then counterstained with Meyer’s hematoxylin (Dako) (2 min), followed by 0.1% calcium bicarbonate bluing reagent (5 min) and coverslipped with aqueous mounting media (Dako).

Following routine paraffin embedding, 5 μ m microtome tissue sections were placed on positively charged glass slides. Tissues were deparaffinized, rehydrated with graded alcohols, and treated with 88% formic acid digestion prior to heat-induced epitope antigen retrieval in the 2100-Retriever^TM (Prestige Medical). Following retrieval, endogenous peroxidase activity was quenched prior to prion epitope detection with anti-prion antibody BAR224 (Caymen chemical) at 2 μ g/mL concentration and Envision+^TM anti-mouse HRP-labeled polymer (Dako). Following antibody treatment, antigen was visualized with 3-amino-9-ethylcarbazole (AEC) substrate chromogen (Dako). Tissue sections were counterstained with Meyer’s hematoxylin (Dako) and 0.1% bicarbonate bluing reagent prior to coverslipping with aqueous mounting media (Dako).

Slides spotted with vaginal cells obtained from swabbed gel applicators were washed in 100%, 95%, and 70% ethanol sequentially, for 5 minutes each. After final washes in dH2O and PBS, slides were blocked with 3% BSA for 30 minutes before adding 1∶100 of a rabbit anti-human cytokeratin 4 primary antibody (Abcam, Cambridge, MA) or 1∶100 of a mouse anti-human cytokeratin 10 (Santa Cruz, Dallas, TX) in blocking buffer for 1 hour. An equivalent amount of normal rabbit or mouse IgG (Santa Cruz, Dallas, TX) was used as a negative control. After two washes in PBS, 1∶200 of a biotinylated goat anti-rabbit or goat anti-mouse IgG secondary antibody (Vector Laboratories, Burlingame, CA) was incubated for 30 minutes. Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) in combination with AEC chromagen (ScyTek Laboratories, Logan, UT) was used for detection according to manufacturers' instructions. Cells were counterstained with hematoxylin before mounting in aqueous mounting media (DAKO, Carpinteria, CA) and coverslipping.