Goat anti chat

Tissues were fixed for 30 min in 4% paraformaldehyde (Electron Microscopy Sciences) and incubated in 0.1 M phosphate buffer (PB) overnight at 4 °C. Fixed retinas were incubated in PBS containing 3% normal donkey serum (blocking agent), 0.05% sodium azide, 0.5% Triton X-100 for 2 h. This was followed by incubation in blocking solution and primary antibody against ChAT (Millipore, AB144P, goat anti-ChAT, 1:500 v/v) for five nights at 4 °C. Afterwards, tissues were rinsed in 0.1 M PB and incubated for two nights at 4 °C with secondary antibody against goat IgG (Jackson ImmunoResearch, 711-605-152, donkey anti-goat Alexa 647, 1:500 v/v) and streptavidin (Thermo Scientific, DyLight 488, 1:500 v/v). Following immunostaining, retinas were mounted on slides with Vectashield Antifade mounting (Vector Labs) medium.

After electrophysiological experiments, some retinas were fixed in fresh 4% paraformaldehyde in PBS at 4 degrees C for 1 hour. After fixation, the retinas were washed and incubated at 4 degrees C with primary antibodies for 4–5 days. Secondary antibody incubation at room temperature for at least 2 hours preceded mounting on a glass slide with spacers, ganglion cell side up, with Prolong Gold (Invitrogen). Whole mount images were obtained on a LSM 710 inverted NLO microscope at 20X or 40X (Zeiss). The primary antibodies used were: anti-GFP (rabbit, Life Technologies; chick, Abcam); mouse anti-Nonphosphoneurofilament H (SMI-32, Covance); goat anti-Osteopontin (R&D Systems); rabbit anti-Parvalbumin, rabbit anti-Calbindin, and mouse anti-Calretinin (all from Swant); anti-vAChT (goat, Promega; guinea pig, Millipore); goat anti-ChAT (Millipore); mouse anti-Brn3a (Millipore); goat anti-Brn3 (raised against Brn3b, Santa Cruz Biotechnology) and mouse anti-Brn3c (Santa Cruz Biotechnology). Dylight405-, Alexa488-, Cy3- and Alexa647-conjugated secondary antibodies were obtained from Jackson Immunoresearch.

Primary antibodies (supplier; dilutions) used in this study were as follows: rabbit anti-Calbindin (Swant; 1∶10,000); goat anti-ChAT (Millipore; 1∶500); rabbit anti-GFAP (DAKO; 1∶8,000); mouse anti-GM130 (BD Transduction; 1∶100); rabbit anti-Hsp25 (Enzo; 1∶8,000); rabbit anti-Iba-1 (Wako; 1∶5,000); rat anti-Ki67 (DAKO; 1∶200); rat anti-Mac2 (Cedarlane;1∶2,000); mouse anti-NeuN (Millipore; 1∶1,000); rabbit anti-p53 (Leica; 1∶1,000). For avidin– biotin–peroxidase immunocytochemistry biotinylated secondary antibodies from Vector Laboratories, diluted 1∶200 were used. FITC-, Cy3-, and Cy5-conjugated secondary antibodies raised in donkey (Jackson ImmunoResearch) diluted at 1∶200 were used for confocal immunofluorescence.

After behavioral analysis, AD11 and WT mice were deeply anaesthetized with chloral hydrate and intracardially perfused with a 4% solution of paraformaldehyde in PBS. Brains were collected and post-fixed in the same solution for 4 h, transferred in 30% sucrose/PBS solution and then sectioned at a sliding freezing microtome (Leica, Wetzlar, Germany). Forty-micrometer sections were collected in 0.05% sodium azide/PBS in 1.5 ml tubes and stored at 4 °C until usage. To detect choline acetyltransferase (ChAT) in basal forebrain neurons, β-amyloid, CD11b in the hippocampus and phosphorylated tau (pTau) in the entorhinal cortex, the following primary antibodies were used: goat anti-ChAT (1:500, Millipore, Billerica, MA); goat anti –NH2 terminus of β-amyloid (1:100; Santa Cruz Biotechnologies, Santa, CA), goat anti-CD11b (1:100, Santa Cruz) and mouse anti-human phosphotau recognizing Ser199 (1:10 clone AT8; Pierce Endogen, Rockford, IL), anti-synaptophysin (1:10, clone SY38, Abcam, Cambridge, UK). The primary antibody signal was detected using the appropriate biotinylated secondary antibody and the avidin horse radish peroxidase system by Vector Laboratories Inc. (Burlingame, CA), with the exception of anti-β-amyloid antibodies that were detected using the avidin-alkaline phosphatase system (Vector Labs).