Cytoseal 60 medium

Brain sections that were rehydrated through 100% and 95 % alcohol to distilled water were stained in 0.1% Cresyl Violet (Acros Organics, #10510-54-0) solution for 5–10 min and then rinsed quickly in distilled water before were differentiated in 95% ethanol for 20 min and dehydrated in 100% ethanol twice for 5 min each. Finally, the sections were cleared twice in Xylene for 5 min and were mounted with Cytoseal 60 Medium (Richard-Allan Scientific, #8310-16). Images were captured under a microscope (Olympus), and 12–15 sections per animal were imaged. The number of surviving neurons in each section was quantified using the ImageJ software (NIH).

Airway sections were formalin fixed and paraffin embedded prior to immunohistochemical staining. Sections were deparaffinized, rehydrated, processed for antigen retrieval and incubated overnight at 4 °C with CREBBP antibody (Table 2). Sections were subsequently incubated with a biotinylated goat anti-rabbit secondary antibody (1:100, Vector Laboratories, Burlingame, CA, USA) prior to visualization with Streptavidin-HRP (Dako) and 3,3-diaminobenzidine (Dako). Slides were counterstained with Harris Hematoxylin solution (Sigma, St. Louis, MO, USA) and dehydrated before coverslipping with Cytoseal 60 medium (Richard-Allan Scientific, Kalamazoo, MI, USA).
Using the Nikon Eclipse 700 (Nikon Instruments, Melville, NY, USA) with a 60× objective and SPOT Advanced software (Diagnostic Instruments, Sterling Heights, MI, USA), five images were obtained from each section. These images were analyzed for positively and negatively stained nuclei using ImagePro Plus software (Media Cybernetics, Rockville, MD, USA).

Thioflavin S staining of protein aggregates in the brain was performed using a previously described method [23 (link)]. Brain sections were incubated with 70% ethanol (Fisher, #A40520) for 1 min and then 80% ethanol for 1 min. Slides were then incubated with 0.1% Thioflavin S (Sigma, #T1892) in 80% ethanol for 15 min. Slides were then rinsed with 80% ethanol for 1 min, 70% ethanol for 1 min, and twice with distilled water. Slides were mounted with Cytoseal 60 Medium (Richard-Allan Scientific, #8310-16), and images were acquired using a fluorescence microscope equipped with the ZEN 2.5 software (Carl Zeiss, Jena, Germany).