Immunostaining was carried out using published methods (Gu et al., 2009 (
link)). S830 sheep anti-HTT antibody (from Dr. G. Bates; 1:1,000 dilution) antibody was used for mHTT aggregate detection;
GFAP antibody (Sigma, 1:20,000 dilution) was used for detection of reactive astrocytes;
NeuN antibody (Millipore, 1:1,000) was used to identify neurons;
ubiquitin antibody (Dako, 1:1,000 dilution) was used for detection of ubiquitin; ACTN2 antibody (Millipore, 1:500) was used to quantify alpha-actinin 2 expression in striatum. For double immunofluorescent staining (e.g. S830/NeuN) we added the S830 antibody and incubated at 4°C for one day, then added NeuN or other antibodies and incubated at 4°C overnight. Images were acquired using a
Dragonfly High-Speed Confocal Microscope 200 and the
Fusion 2.0 software package (Andor, Oxford Instrument). Laser and detector settings were maintained at constant for the acquisition of each immunostaining. For all analyses, at least three images were taken per brain region and slide using 360 oil objective lens, at 2,048 × 2,048 pixel resolution, with z-step size of 1 μm at 10 μm thickness.
Gu X., Richman J., Langfelder P., Wang N., Zhang S., Bañez-Coronel M., Wang H.B., Yang L., Ramanathan L., Deng L., Park C.S., Choi C.R., Cantle J.P., Gao F., Gray M., Coppola G., Bates G.P., Ranum L.P., Horvath S., Colwell C.S, & Yang X.W. (2022). Uninterrupted CAG repeat drives striatum-selective transcriptionopathy and nuclear pathogenesis in human Huntingtin BAC mice. Neuron, 110(7), 1173-1192.e7.
