Mab5374

For western blotting, primary antibodies against Lamp2b (rat; Developmental Studies Hybridoma Bank ABL-93; AB-2134767; 1:1000), CD9 (rabbit; Abcam ab92726; 1:1000), Calnexin (rabbit; StressGen ADI-SPA-860; 1:2000), GFP (mouse; Clontech 632381; 1:5000), mRFP/mCherry (rabbit; Abcam ab167453; 1:500) and DNAJB6 (rabbit; housemade; 1:2000) were used. Odyssey secondary anti-mouse, -rabbit and -rat antibodies (Li-COR, LI 926-68070, LI 926-32211 and LI 926-32219) were used at 1:5000 dilution. For immunocytochemistry, CD9 (1:100) was used followed by staining with secondary antibodies conjugated with Alexa 546 (goat; Invitrogen A-11010; 1:500). Antibodies for immunohistochemistry included EM48 (mouse; Millipore MAB5374; 1:200) and anti-mouse conjugated with biotin (horse; Vector BA9200; 1:300).

Animals were killed using sodium pentobarbital and perfused transcardially with saline followed by 4% paraformaldehyde, and their brains were processed for immunocytochemistry as previously described. Sagittal equidistant cryosections of sections (20 μm) spanning the whole brain were analysed. Human cells were identified through immunostaining with anti-human nuclear antigen (1:800, MAB1281, Millipore, Temecula, CA, USA). Engrafted human cells were mapped using Metamorph imaging software and an automated fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Brain sections were then co-stained to define phenotype, using combinations of the following antibodies: mouse anti-huntingtin antibody clone EM48 (1:250, MAB5374, Millipore); mouse anti-glial fibrillary acidic protein (GFAP) (1:800, SMI-21, Covance, Princeton, NJ, USA); rabbit anti-Olig2 (1:400, RA25081 Neuromics, Edina, MN, USA); rabbit anti-PDGFRα (1:400, 5241 Cell Signaling Technology, Danvers, MA, USA). Slides were analysed serially every twenty-fourth section using the optical fractionator method to estimate the total number of engrafted human cells of each histological marker (GFAP, Olig2, PDGFRα) using StereoInvestigator imaging software (MicroBrightField, Burlington, VT, USA).

Coronal brain sections of 50 μm thickness were cut with an electronic microtome (Microm HM 650 V). The slides from bregma −0.60 to bregma −0.80 mm were used for Hematoxylin/eosin staining and immunostained with monoclonal antibody anti-Huntingtin (MAB5374 – Millipore – 1:1000) or polyclonal anti-GFAP (AB5804 – Millipore – 1:1000 [33 (link)]). For each animal, systematic pictures were taken with the slide scanner Hamamatsu nanozoomer. The areas of lateral ventricles and cortex thickness were measured with NDP software (Nanozoomer Digital Pathology Virtual SlideViewer). Quantifications of aggregates number and size, or GFAP signal, were scored with ImageJ software from 256 μm × 256 μm images. Representative pictures for these immunostainings obtained with the confocal laser scanning microscope model LSM780 (Carl Zeiss) are shown.
TAMRA-P42-TAT injected mice were also immunostained with rabbit monoclonal anti-DARPP32 (MAb2306 - Cell Signaling Technology - 1: 1600), secondary detected by Alexa 488 anti-rabbit (1: 2000) to label the striatum, and with mouse monoclonal anti-NeuN (MAB377 – Millipore - 1: 1000), secondary detected by Dye light blue anti-mouse (1: 500), as a neuronal marker.

Tissue sections from all animals were rinsed in Tris-buffered saline (TBS) with 0.05% Triton X-100 before being blocked in TBS solution containing 10% normal goat serum (Gibco). Sections were incubated in an em48 (Millipore, MAB 5374, 1:500) or 1–82 aa/2B4 (MAB 5392, 1:500) primary antibody solution overnight. Sections were washed and incubated in a goat anti-mouse biotinylated secondary antibody solution for 1 hr (Vector Labs, BA-9200, 1:500). Tissue was rinsed and the signal was developed using a standard Vectastain ABC kit (Vector Laboratories, PK6100) for 1 hr before developed in 3,3′-diaminobenzidene (DAB) with nickel (II) sulfate (0.05% DAB, 2.5% nickel (II) sulfate hexahydrate, 0.02% H₂O₂, Tris-buffered saline).