Avidin biotin complex method

Mouse brains were prepared for immunohistochemistry. Serial 6-μm thick sections were deparaffinized, hydrated, and blocked in 2% fetal bovine serum before incubation with primary antibody overnight at 4 °C. Sections were incubated overnight at 4 °C with primary antibodies Aβ-4G8 (1:150 Covance), HT7 (1:150), AT8 (1:50), 4-HNE (1:20; ab48506,) then incubated with secondary antibody and developed using the avidin–biotin complex method (Vector Laboratories, Burlingame, CA) with 3,3′diaminobenzidine as chromogen. 4G8 antibody is reactive to amino-acid residues 17–24 of Aβ and the epitope lies within amino acids 18–22 of Aβ. 4G8 Aβ antibody reacts to abnormally processed isoforms, as well as precursors. The epitope for HT7 lies within amino acids 159–163 of tau.

MAP-2 staining was performed on pups' coronal brain sections according to the protocol previously published [33 (link)]. Briefly, after antigen retrieval with citrate buffer, endogenous peroxidase activity was blocked, followed by second blocking and incubation with MAP-2 at a dilution of 1: 1000 (mouse monoclonal antibody ; Sigma, St. Louis, MI, USA). B iotinylated goat anti-mouse immunoglobulin G (Vector, Burlingame, CA, USA) addition was followed by the antibody detection by using the avidin-biotin complex method (Vector), with 3,3-diaminobenzidine (DAB).
The infarct area corresponds to a loss of MAP-2 staining. 10 samples from each study group of both study designs were analyzed as in our previous study [33 (link)]. The ratio of the MAP-2 positive area in sham operated animals was considered as 1.0 by default.

Immunostaining was performed as described in our previous studies29 (link)33 (link). Briefly, serial coronal sections were mounted on 3-aminopropyl triethoxysilane (APES)-coated slides. Every eighth section from the habenular to the posterior commissure (6–8 sections per animal) was examined using unbiased stereological principles. The sections for testing Aβ were deparaffinized, hydrated, pretreated with formic acid (88%) and subsequently with 3% H₂O₂ in methanol. The sections used for testing HT7, AT8, AT180, AT270, synaptophysin, PSD95 and MAP2 were deparaffinized, hydrated and subsequently treated with 3% H₂O₂ in methanol, and then antigen retrieved with 10 mM sodium citrate buffer. Sections were blocked in 2% fetal bovine serum before incubation with the appropriate primary antibody overnight at 4 °C. After washing, sections were incubated with biotinylated anti-mouse IgG (Vector Lab, Burlingame, CA) and then developed by using the avidin-biotin complex method (Vector Lab, Burlingame, CA) with 3,3′-diaminobenzidine (DAB) as a chromogen. Light microscopic images were captured using software QCapture 2.9.13 (Quantitation Imaging Corporation, Surrey, Canada) with the auto-exposure option. These images were used to calculate the area occupied the immunoreactivities using the software Image-ProPlus (Media Cybernetics).

After behavioral testing, three out of six rats from the normal group, three out of the seven rats from the lesion group, three out of seven rats from the implantation group, two out of five rats from the simulation group were perfused with cold 4 % paraformaldehyde in phosphate buffer saline (pH 7.2). Their brains were removed, post-fixed, and transferred to 30 % sucrose. The brains were cut into 30 μm coronal sections using a freezing microtome. The cryoprotection solution consisted of 0.1 M phosphate buffer (pH 7.2), 30 % sucrose, 1 % polyvinylpyrrolidone, and 30 % ethylene glycol. Tissue was stained with cresyl violet to confirm correct placement of the needle tracks. To detect cholinergic cells, tissue was immunohistochemically processed using polyclonal antibodies against choline acetyltransferase (ChAT; 1:100; cat# AB144P, Chemicon). The sections were stained using the avidin-biotin complex method (Vector Labs, Burlingame, CA, USA) with diaminobenzidinetetrahydrochloride as the substrate. Also fluorescence immunohistochemistry was performed with ChAT primary antibody (1:50; cat# AB144P, Chemicon) and Texas Red (1:400; cat# ab6883, abcam). Anatomical landmarks from a stereotaxic atlas [24 ] were used to localize the medial septum (MS).