Computational protocol: Tagging of Endogenous BK Channels with a Fluorogen-Activating Peptide Reveals β4-Mediated Control of Channel Clustering in Cerebellum

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Protocol publication

[…] Lipofuscin-like fluorescence was identified by detection with 670 long-pass under 488 or 514 nm excitation due to its broad excitation and emission spectra. Lipofuscin fluorescence was masked in all channels prior to analysis using Imaris for 3D images and Fiji for 2D images. Deconvolution when applied was performed using a 3D-blind algorithm in NIS Elements (Nikon). Low dynamic range channels were smoothed with a 3 × 3 median or Gaussian filter of the approximate point-spread function half width (0.232 μm, approximately one pixel radius).Purkinje cell dendritic fluorescence was measured in 2D sections by measuring mean molecular layer signal normalized to granule layer signal (n = 10 fields from 1 WT, 16 fields from 2 FAP-BKα Het, 18 fields from 2 FAP-BKα Hom mice). PC soma fluorescence was measured in 2D optical sections using ImageJ by calculated total cell fluorescence (CTCF), whereBackground was measured by taking ROIs in the granule cell layer, which lacks BKα. For analysis, 16 somata from 1 WT, 17 somata from 2 FAP-BKα Het, 17 somata from 2 FAP-BKα Hom mice were used.Analysis of PC puncta brightness and abundance was performed using a semi-automated algorithm using the spots function of Imaris. For β4 experiments, images were acquired from lobes 7 and 8 of the cerebellum of FAP-BKα Het mice. Because Imaris regions of interest are cubic but PC somata are not, identified spots outside the cell of interest were excluded from analysis. Image IDs were blinded for analysis. […]

Pipeline specifications

Software tools Imaris, ImageJ
Application Microscopic phenotype analysis
Organisms Mus musculus
Diseases Cerebellar Diseases, Epilepsy, Infertility