PLA Assay for BIN1-LVGCC-β1 Interaction

PLA was adapted from Rush et al., 2020 (link). Briefly, neurons on coverslips were fixed and permeabilized as with ICC, then were incubated overnight with primary antibodies for BIN1 (Santa Cruz, sc-30099, 1:500) and LVGCC-β1 (Abcam, S7-18, 1:1,000) overnight at 4°C, then PLA was performed using the Duolink In Situ Fluorescence kit (Sigma, DUO92004-100RXN). After PLA, coverslips were incubated with secondary antibody to view BIN1 and mounted with Duolink In Situ Mounting Medium with DAPI. Fluorescent images were taken using an epifluorescence microscope at 60x with four channels: DAPI (nuclei), FITC (PLA), and TRITC (mKate2). 7–9 images per slide were obtained and analyzed using ImageJ (v. 2.0.0-rc-69/1.52 p). PLA puncta were quantified using ImageJ particle analyzer, and the average number of puncta per field of view (FOV) or each coverslip was used for analysis.

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Voskobiynyk Y., Roth J.R., Cochran J.N., Rush T., Carullo N.V., Mesina J.S., Waqas M., Vollmer R.M., Day J.J., McMahon L.L, & Roberson E.D. (2020). Alzheimer’s disease risk gene BIN1 induces Tau-dependent network hyperexcitability. eLife, 9, e57354.

Publication 2020

sc-30099 S7-18 Duolink In Situ Fluorescence kit

Antibodies Antibody Dapi Fitc Fluorescence Microscope Neurons Nuclei Tritc

Corresponding Organization :

Other organizations : University of Alabama at Birmingham

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Variable analysis

independent variables

Primary antibodies for BIN1 (Santa Cruz, sc-30099, 1:500) and LVGCC-β1 (Abcam, S7-18, 1:1,000)

dependent variables

Number of PLA puncta per field of view (FOV) or each coverslip

control variables

Neurons on coverslips were fixed and permeabilized as with ICC
Primary antibodies were incubated overnight at 4°C
PLA was performed using the Duolink In Situ Fluorescence kit (Sigma, DUO92004-100RXN)
Coverslips were incubated with secondary antibody to view BIN1 and mounted with Duolink In Situ Mounting Medium with DAPI
Fluorescent images were taken using an epifluorescence microscope at 60x with four channels: DAPI (nuclei), FITC (PLA), and TRITC (mKate2)
7–9 images per slide were obtained and analyzed using ImageJ (v. 2.0.0-rc-69/1.52 p)

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