Quantifying Huntingtin Aggregates in MSNs

Medium spiny neuron (MSN) specific mutant HTT aggregates were assessed using a multiplexed immunofluorescence approach staining for HTT protein (EM48 antibody, cat.# MAB5374, Millipore-Sigma) and anti-DARPP-32 (19A3 antibody, cat.# 2306S, New England Biolabs - expressed almost exclusively by striatal medium spiny neurons), as described^{140 (link)}. After rehydration, slides were subjected to antigen retrieval in 10 mM sodium citrate for 20 min in a steamer and cooled to room temperature for 1 h. Slides were washed twice in 1x PBS + 0.05% Tween-20 (1x PBST) (2 min, 2 times). Slides were blocked in 10% normal goat serum for 1 h at room temperature and incubated with primary antibody overnight at 4 °C. Secondary antibody incubation was performed for 1 h at room temperature. Slides were mounted with Hardset VectaShield with DAPI. Aggregates were then blindly assessed in two ways:

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Nakamori M., Panigrahi G.B., Lanni S., Gall-Duncan T., Hayakawa H., Tanaka H., Luo J., Otabe T., Li J., Sakata A., Caron M.C., Joshi N., Prasolava T., Chiang K., Masson J.Y., Wold M.S., Wang X., Lee M.Y., Huddleston J., Munson K.M., Davidson S., Layeghifard M., Edward L.M., Gallon R., Santibanez-Koref M., Murata A., Takahashi M.P., Eichler E.E., Shlien A., Nakatani K., Mochizuki H, & Pearson C.E. (2020). Slipped-CAG DNA binding small molecule induces trinucleotide repeat contractions in vivo. Nature genetics, 52(2), 146-159.

Publication 2020

MAB5374

A protein Antibody Antigen Dapi Darpp 32 Goat Immunofluorescence Medium spiny neurons Rehydration Serum Sodium citrate Striatal Tween 20

Corresponding Organization :

Other organizations : Osaka University, Hospital for Sick Children, University of Toronto, University of Iowa, New York Medical College, University of Washington, Newcastle University

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Variable analysis

independent variables

Mutant HTT aggregates in medium spiny neurons (MSNs)

dependent variables

Assessment of mutant HTT aggregates using multiplexed immunofluorescence

control variables

Slides were subjected to antigen retrieval in 10 mM sodium citrate for 20 min in a steamer and cooled to room temperature for 1 h
Slides were washed twice in 1x PBS + 0.05% Tween-20 (1x PBST) (2 min, 2 times)
Slides were blocked in 10% normal goat serum for 1 h at room temperature
Primary antibody incubation was performed overnight at 4 °C
Secondary antibody incubation was performed for 1 h at room temperature
Slides were mounted with Hardset VectaShield with DAPI

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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