Citiflour

Cultured WT or KLHL1-KO hippocampal neurons were fixed at 11 DIV with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) following standard methods (Perissinotti et al., 2014 (link)). Primary antibodies were diluted in blocking solution containing 2% goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) plus 0.4% saponin in PBS and incubated overnight at 4°C (MAP2, 1:1000, EnCor Biotechnology, FL; synapsin I, 1:1000, Millipore, CA; gephyrin, 1:500, SySy, Germany; GAD67 1:500, Thermo Scientific; PSD-95, 1:1000, NeuroMab; VGlut-1, 1:1000, SySy; synapsin I, 1:1000, Millipore, CA). Samples were incubated in alexa fluor-conjugated secondary antibodies (1:2,000; Life Technologies) for 1 h at room temperature. Coverslips were mounted on slides with Citiflour (Ted Pella, Redding, CA) and stored at −20°C for subsequent detection (Multiphoton Leica TCS SP5). Data was analyzed with ImageJ freeware (NIH) (Rasband, 1997–2006 ). Images were thresholded using the Otsu plugin. Synaptic puncta number was measured in n sample areas = 246 μm² each. The JACoP plugin was used to calculate Mander’s correlation coefficients (co-localization percentages), which provides a good quantification of signal co-localization between samples of different intensities (Otsu, 1979 (link); Bolte and Cordelières, 2006 (link)).

To perform immunostaining, Xenopus muscle cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, rinsed 3 times with PBS, and then permeabilized with 0.1% Triton X-100 in PBS for 10 min. After blocking with 5% bovine serum albumin (BSA, Sigma) for at least 1 h, the cells were incubated with primary antibodies for 2 h and then with Alexa 488-conjugated secondary antibodies (1∶400 dilution; Life Technologies) for 45 min. After extensive washing in PBS, the coverslips were mounted on glass slides with an anti-bleaching agent (Citiflour, Ted Pella).