Computational protocol: Frequency of nuclear mutant huntingtin inclusion formation in neurons and glia is cell‐type‐specific

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Protocol publication

[…] To determine the nuclear inclusion proportion per cell type, images were taken with a Leica SP8 confocal microscope using a 63× oil objective and Leica LAS X imaging software. A minimum of five images was taken from each sample, the number of cells differed per cell type ranging from 3 to 50 cells per field. Per image, the total number of cell‐marker‐positive cells and the number of cells with inclusions were counted. Per cell type a minimum of 200 cells were included.To quantify the number of cells in HD mice and their wild‐type littermates, images were acquired using a Leica DM RA microscope equipped with a Plan Apo 25× 1.40 oil objective (Carl Zeiss), a cooled charge‐coupled device camera (KX1400; Apogee Instruments, Logan, UT, USA), and Image‐Pro Plus software. DAPI‐stained nuclei were counted by a custom‐made Matlab script. With this script, the total nuclear surface area was measured and the number of distinct objects was counted. The number of positive cells was counted manually by persons that were unaware of the mouse genotypes. [...] One hundred micrograms of protein suspension in 300 µL of 15% SDS was blotted onto a cellulose acetate membrane using a vacuum applicator. Blots were washed twice with 0.2% SDS and fixed in 0.5% glutaraldehyde. Blots were blocked with 4% nonfat milk (Nutricia, Schiphol, The Netherlands) in Tris‐buffered saline with Tween‐20 and probed with primary anti huntingtin antibody 3702‐1 (Epitomics, Burlinggame CA, USA), and secondary antibody horse radish peroxidase conjugated goat‐anti‐rabbit (Santa Cruz). Blots were visualized with enhanced chemoluminescence (ECL, #32106, ThermoFisher) and Hyperfilm ECL (#28906837, GE healthcare, Little Chalfond, United Kingdom). Signal intensity was quantified with imageJ‐software. […]

Pipeline specifications

Software tools LAS X, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Huntington Disease, Nervous System Diseases, Heredodegenerative Disorders, Nervous System