Histology and immunohistochemistry
Mice were anesthetized with ketamine-xylazine before intracardiac perfusion with 4% paraformaldehyde in PBS. Brains were removed and kept in the fixative for 12 - 16 h (40 C). Forty μm coronal sections were prepared with a vibratome (Leica VT1000S) and stored in cryoprotectant solution at -200 C before use. Paraffin sections from the blocks of autopsy AxD human brains were deparaffinized and were used for immunostaining. Some were stained with H&E.
Primary antibodies were used against: (i) glial fibrillary acidic protein (GFAP): mouse monoclonal (1:1000, G3893, Sigma-Aldrich, St. Louis, MO), rabbit polyclonal (1:1000, Z 0334, Dako, Carpinteria, CA); (ii) CD44: rat monoclonal (1:200, #217594, Millipore); (iii) Ki67: mouse monoclonal (1:100, #550609, BD Pharmingen, San Jose, CA); (iv) a centrosome marker, pericentrin: rabbit polyclonal (1:400, PRB-432C, Covance). Secondary antibodies included: anti-mouse Alexa Fluor 488 and 594; anti-rabbit Alexa Fluor 488 and 594; anti-rat Alexa Fluor 488 and 594; all from goat or donkey (1:300, Molecular Probes, Eugene, OR).
For immunofluorescence, after blocking with 10% normal goat (or donkey) serum (30 min, at room temperature (RT)), free-floating sections were incubated in a mixture of primary antibodies raised in different species for overnight (40 C). For visualization, Alexa Fluor-conjugated secondary antibodies were applied for 1 h at RT. For visualization of nuclei Fluorescent Nissl reagent (NeuroTrace 640/660 deep-red, 1:150, Molecular Probes, 30 min after secondary antibodies, RT) and DAPI (5 μg/ml; D9542, Sigma-Aldrich, added to secondary antibodies for the last 10 min of incubation) were used. Paraffin sections and mouse brain sections were treated with Antigen Unmasking Solution (Vector Laboratories, # H-3300, Burlingame, CA) according to manufacturer’s recommendations before blocking with serum. Blocking serum, primary, secondary antibodies, and DAPI were applied in 0.2% Triton X-100 in PBS. Sections for fluorescent microscopy were mounted on slides in Vectashield (Vector Lab). To control for the specificity of immunostaining, primary antibodies were omitted and substituted with appropriate normal serum. Slides were viewed using a Nikon A1R MP confocal microscope. 3D reconstructions were generated from stacks of images with confocal microscope software NIS-Elements.
Fluoro-Jade B Staining was performed according to manufacturer’s recommendations (Millipore). Briefly, 40 μm coronal brain slices were mounted on the slides, dried, exposed to 1% NaOH in 80% alcohol for 5 min, followed by 70% alcohol (2 min) and distilled water (2 min). After pretreatment in 0.006% potassium permanganate (15 min RT) followed by washing (distilled water, 2 min), slides were incubated in 0.0004% of FluoroJade B (in distilled water with 0.1% acetic acid) for 20 min (RT) and after drying were coverslipped with DPX (Sigma-Aldrich). This method was initially proposed and is widely used for the detection of degenerating neurons [
22]. Reactive astrocytes [
3] and Rosenthal fibers [
30] were also stained with Fluoro Jade B.
Electron microscopy (EM)
For regular transmission EM vibratome slices were additionally fixed in 2.5% glutaraldehyde in PBS (several hrs at 4 °C), postfixed in 2% osmium tetroxide in 0.2 PBS (2 h at 4 °C), dehydrated and flat-embedded in Epon-Araldite (Electron Microscopy Sciences, Hatfield, PA) on ACLAR Embedding Film (Ted Pella, Inc., Redding, CA). Areas of interest were identified under light microscope, cut from sections, and glued onto resin blocks. Ultrathin sections were cut with Reichert Ultracut E, stained with uranyl acetate and lead citrate, and examined with a JEOL 1200 electron microscope.
For postembedding immunogold immunocytochemistry small pieces of the neocortex and hippocampus were embedded in LR White Resin (Berkshire, England). Ultrathin sections on nickel grids were blocked with 10% donkey serum (30 min, RT), incubated with primary antibodies anti-GFAP (rabbit polyclonal, 1:200, Dako, for single staining, and mouse monoclonal, Sigma, 1:100), for double immunostaining with anti-alphaB-crystallin (rabbit polyclonal, 1:100, Millipore) applied overnight at 40 C, followed by secondary antibodies conjugated with gold particles (12 nm in diameter for GFAP, and 18 nm for alphaB-crystallin) (all at 1:50) for 1.5 h at RT. Blocking donkey serum, primary and secondary antibodies were applied in PBS and grids were put on the top of droplets of corresponding solutions. Ultrathin sections were stained with uranyl acetate and examined with a JEOL 1200 electron microscope.
Quantitative analysis
The numbers of RFs and the area they occupied were counted in the images (merged from stacks of 6 adjacent images with 1024 × 1024 pixel resolution, observed area 295 × 295 μm, captured by confocal microscopy at a distance of 0.5 μm from each other) obtained from neocortices in coronal sections stained with Fluoro-Jade B (10 images from each section, 5 sections per animal). Images were transferred to Image J 1.46r (public domain), grayscaled and quantified based on the optical density (OD). The size of small RFs, detected with the electron microscope, and the numbers of RFs with different shapes were determined in EM images. We counted RFs in all parts of astrocytes, including cell bodies, processes, and endfeet.