Immunostaining Protocol for GRP78, TDP43, BMAL1, and GFP

Rabbit anti-GRP78 (also called BiP; 1:4000, Novus Biologicals, San Diego, CA, United States), monoclonal mouse anti-TDP43 (1:32,000, Abcam, San Francisco, CA, United States), guinea pig anti-BMAL1 (1:15,000, Millipore, Billerica, MA, United States), and sheep anti-GFP (1:3,000, AbD Serotec, Raleigh, NC, United States) were used. Species appropriate, fluorophore-conjugated, minimally cross reactive secondary antibodies were obtained from Jackson Immuno (West Grove, PA, United States) and used at a concentration of 1:500. Specificity of anti-BiP antibody was previously verified via siRNA knockdown (Kitahara et al., 2011 (link); Maddalo et al., 2012 (link)). Specificity of anti-TDP43 antibody was previously verified via shRNA knockdown (Lee et al., 2015 (link)). Specificity of anti-Bmal1 antibody was verified in Bmal^−/− tissue (LeSauter et al., 2012 (link)).

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Henry F.E., Sugino K., Tozer A., Branco T, & Sternson S.M. (2015). Cell type-specific transcriptomics of hypothalamic energy-sensing neuron responses to weight-loss. eLife, 4, e09800.

Publication 2015

Rabbit anti-GRP78 sheep anti-GFP

Anti antibody Antibodies Antibody specificity Biologicals Cross reactive Grp78 Guinea pig Mouse Novus Rabbit Sheep Shrna Sirna Tdp43 Tissue

Corresponding Organization :

Other organizations : Janelia Research Campus, Howard Hughes Medical Institute, MRC Laboratory of Molecular Biology, Medical Research Council

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Variable analysis

independent variables

Antibody concentration for rabbit anti-GRP78 (1:4000)
Antibody concentration for monoclonal mouse anti-TDP43 (1:32,000)
Antibody concentration for guinea pig anti-BMAL1 (1:15,000)
Antibody concentration for sheep anti-GFP (1:3,000)

dependent variables

Not explicitly mentioned

control variables

Species appropriate, fluorophore-conjugated, minimally cross reactive secondary antibodies used at a concentration of 1:500

controls

Positive control: Specificity of anti-BiP antibody previously verified via siRNA knockdown (Kitahara et al., 2011; Maddalo et al., 2012)
Positive control: Specificity of anti-TDP43 antibody previously verified via shRNA knockdown (Lee et al., 2015)
Positive control: Specificity of anti-Bmal1 antibody verified in Bmal−/− tissue (LeSauter et al., 2012)

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