Sig 39300

For histological analysis, the tissues used were fixed with 4% paraformaldehyde, and serially frozen coronal brain sections (40 μm) were obtained as described [12 (link)]. To detect Ach or 5-HT fibers and neurons and amyloid deposits in the same brain section, a serial double-immunocytochemical staining procedure was employed. Free-floating frozen sections were first immunodetected with an anti-ChAT antiserum (AB143, MilliporeSigma, Burlington, MA, USA) or anti-5-HT antiserum (Immunostar, Hudson, WI, USA). Sections were then incubated with the appropriate 2nd antibody, followed by the ABC method (Vector Laboratories, Newark, CA, USA), using diaminobenzidine–nickel (DAB-Ni) as the chromogen to obtain the dark blue/black reaction products. The sections were then immunoreacted with anti-Aβ mouse monoclonal antibodies (6E10, Covance, SIG-39300, Burlington, NC, USA), and DAB was used as the chromogen to obtain brown color products. We also used another anti-Aβ mouse monoclonal antibody (4G8, Covance, SIG-39200, Burlington, NC, USA) for the detection of amyloid pathology.

For senile plaques staining, coronal sections (12 μm) were cut using a Leica cryostat (Leica CM1850). Primary 6E10 antibody (SIG-39300, Covance, Princeton, NJ, USA) was used on the sections overnight at 4°C. Sections were incubated with biotinylated secondary antibody (AK-6602, Vector) for 45 min. Sections were developed using the ABC elite kit (AK-6600, Vector). Image-Pro plus software (Media cybernetics, USA) was used to measure and recorded as the average plaque areas per field. Six slices per mouse were used to count the plaque number in a blinded manner.

Brain dissections were performed as described previously (Costa et al., 2011 (link)). Aβ plaques in Drosophila brains were quantified by using ImageJ (NIH). Brain plaque quantification was only undertaken on images that were acquired under identical conditions and on the same day. At least four different brain samples were quantified for each genotype. Differences in plaque counts were tested for significance using two-way ANOVA. To probe whether Aβ deposits were intracellular or extracellular, Drosophila brains were stained with TOTO-3 (Life Technologies) for nuclear staining and with phalloidin (Life Technologies) to label intracellular actin. The presence of Aβ deposits was detected with the 6E10 monoclonal antibody (Covance, SIG-39300, 1:100 dilution). Aβ deposits were assessed across consecutive 1 µm confocal slices to determine whether they cross or remain within cellular boundaries.

Briefly, hemibrains were cut into paraffin sections (4 μm), followed by dewaxing and rehydration. Then, sections were boiled for 15 min in citrate buffer (10 mM) at pH 6.0 by heating in a microwave and cooled naturally. Next, they were incubated with H₂O₂ (3% in PBS) for 10 min to inhibit endogenous peroxidases and blocked for 1 h with blocking buffer (5% BSA) at room temperature, followed by incubation with a mouse monoclonal anti‐β‐Amyloid 6E10 (1:1000; SIG‐39300, Covance) overnight at 4°C. On the next day, sections were incubated with a secondary antibody (HRP‐conjugated goat anti‐mouse IgG, Protech) for 1 h at 37°C and finally reacted with DAB (Zhongshan Golden Bridge) and stained by hematoxylin. Images were captured using a light microscope (IX71, Olympus). For the hippocampal or cortical region, a photo of the entire hippocampus or cortex of each brain was captured, and the number and area of Aβ plaques were counted. Four hemibrains were involved in each group. The number and area of Aβ plaques in the cortex and hippocampus were counted in a blinded manner using Image‐Pro Plus software.

Immunofluorescence was performed as described earlier with some modifications [31 ]. Briefly, brain sections were degreased in acetone for 20 min at 4 °C. After incubated with a blocking solution (10% normal goat serum, 0.3% Triton X-100 in PBS) at room temperature for 1 h, the sections were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: mouse anti-6E10 monoclonal antibody (1:500; SIG-39300; Covance, Princeton, NJ), mouse anti-12F4 monoclonal antibody (1:500; SIG-39144; Covance), and rabbit anti-Iba-1 polyclonal antibody (1:200; 019-19741; Wako Chemicals, Japan). After rinsing in PBS, sections were incubated with secondary antibodies: Alexa Fluor-488-conjugated goat anti-rabbit IgG antibody (1:300; R11034; Invitrogen, Carlsbad, CA) and Alexa Fluor-594-conjugated goat anti-mouse IgG antibody (1:500; R11005; Invitrogen). The fluorescent imaging was visualized by using Olympus IX-81-inverted fluorescence microscope and FV1000 confocal laser scanning microscope (Olympus Corporation, Japan).

Coronal cryosections (20 μm) of perfused mouse brains were used for immunohistochemistry. Sections obtained were stored in 0.1% NaN₃ and PBS in a cold room. For immunohistochemistry, sections were treated with 50% methanol for 15 min. Then, sections were washed three times for 5 min in PBS and blocked in 3% BSA, 0.1% Triton X‐100, and PBS (blocking buffer) for 30 min, followed by overnight incubation with the primary antibody in blocking buffer. Next, sections were washed three times in 0.1% Triton X‐100 and PBS and incubated with Alexa Fluor 488‐conjugated or Alexa Fluor 594‐conjugated secondary antibodies (1:500; Invitrogen) for 90 min, washed three times with 0.1% Triton X‐100 and PBS for 5 min. Finally, the sections were mounted on glasses in tap water and embedded. The following primary antibodies were used with respective concentrations: rabbit anti‐ionized calcium‐binding adapter molecule 1 (Iba‐1; 1:500; 019–19741, Wako), and rat anti‐CD68 (1: 400; MCA1957, Bio‐Rad), mouse anti 6E10 (1:500; SIG‐39300, Covance). Fluorescence microscopy was done on an BX61 equipped with a disk‐spinning unit (Olympus) or an A1‐MP (Nikon) laser‐scanning microscope, and images were processed in Cell‐P (Olympus) or NIS elements (Nikon).