*4.7. Antibodies and Immunochemicals for Immunohistochemistry*

The primary antibodies used in this study were: rabbit polyclonal anti-Iba1 (1:100; Wako Chemicals USA, Inc., Los Angeles, CA, USA), mouse monoclonal anti-5-bromodeoxyuridine (BrdU, 1:25; DakoCytomation, Glostrup, Denmark), and mouse monoclonal anti-Aβ (6E10, 1:100; Covance, Emeryville, CA, USA). The secondary antibodies used were: Alexa Fluor-568 goat anti-mouse IgG, and Alexa Fluor-488 goat anti-rabbit IgG (1:400; both from Molecular Probes; Eugene, OR, USA), fluorescein-conjugated goat anti-mouse IgG (1:25; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA).

#### *4.8. Tissue Processing, Immunohistochemistry and Morphometric Analyses*

Four weeks after the beginning of LGF or saline treatment transgenic mice were perfused intracardially under deep anesthesia with 10 mL of heparinized isotonic saline, followed by 40 mL of 4% paraformaldehyde. Brains were postfixed in the same solution for 24 h at 4 ◦C, cryoprotected and frozen, before sectioning into 20 μm-thick coronal sections in a cryostat. For quantitative measurements and immunohistochemical analysis of the dorsal hippocampus and dorsal cortex, coronal sections were obtained at the antero-posterior level of −2.5 to −3 mm from Bregma [84].

Tissue sections were mounted on coated FLEX IHC microscope slides (DakoCytomation, Glostrup, Denmark), treated with sodium acetate 10 mM, pH 6.0, at 95 ◦C for 4 min, and blocked with 5% normal goat serum (NGS) and 0.1% Triton-X 100 in phosphate buffer saline (PBS), pH 7.4, for 30 min. Primary antibodies diluted in 0.5% NGS in PBS, were applied for 24 h at 4 ◦C, and were visualized using fluorescent secondary anti-rabbit and anti-mouse antibodies diluted in 0.5% NGS in PBS. The slides were coverslipped in a medium containing p-phenylenediamine and bisbenzimide (Hoechst 33342; Sigma, St. Louis, MO, USA) for detection of nuclei. For double immunolabeling with anti-Iba1 and anti-BrdU antibodies, the former immunostaining was performed prior to the immunodetection of BrdU incorporated into nuclei. The latter detection needed pretreatment of sections with 2N HCl at 37◦C for 30 min, previous to the NGS blocking step. In the case of Aβ immunodetection in plaques, sections were pretreated with 70% formic acid for 20 min at RT, followed by the blocking step.

For quantification of proliferating BrdU-positive cells, microglial Iba1-positive cells, and Aβ 6E10-positive plaques, panoramic views were obtained from one representative coronal section for each immunostaining technique and for each brain. These panoramic views, where the complete transversal surface of dorsal hippocampus and dorsal cortex of each cerebral hemisphere were visualized (3 mm<sup>2</sup> for each structure), were obtained by using the stitching tool of the software NIS ELEMENTS C, version BR 3.2 (Nikon Instruments Inc., Tokio, Japan), coupled to a Nikon ECLIPSE Ti-e microscope (Nikon Instruments Inc., Tokio, Japan) with a motorized stage, and the 10× objective. Four WT, 4 APP and 4 APPL-GF were analyzed for these evaluations. The data entered in the corresponding graphs were: (a) the mean ± SEM obtained from the data of both cerebral hemispheres (number of plaques/mm2); (b) the mean of 2 different regions in the cortex and of 4 different regions in the hippocampus of one cerebral hemisphere (density of Iba1-positive cells); and (c) the sum of data from both hemispheres (density of BrdU-positive cells).

#### *4.9. Brain Regions and Tissue Preparation for Biochemical Analysis*

After decapitation the brain was extracted and the hippocampus and cortex were free-hand dissected, then the samples were frozen on dry ice for biochemical studies (Western blot). For the protein extraction, dried tissue samples were weighed and placed in six volumes (w/v) of phosphate-buffered saline (PBS) with protease inhibitor cocktail 1× (Calbiochem, Temecula, CA, USA) and 20 mM N-ethylmaleimide to inactivate deubiquitinating enzymes and subjected to two 30-s rounds of sonication. The lysates were immediately boiled for 5 min and centrifuged at 12,000× *g* at 4 ◦C for 30 min. The supernatant-PBS-inhibitors was defined as the soluble fraction and was used for protein analysis by Western blot. To obtain the total Aβ (soluble plus insoluble) fraction, the initial homogenate (200 μL) was extracted with 5 M guanidine in 6 mM Tris–HCl, pH 8.0 by rotating the sample at room temperature overnight. The sample obtained after guanidine extraction represented the total fraction. Soluble and total fractions were used for protein analysis by Western blot.
