The Journal of Neuroscience, November 24, 2004, ():
Regulation of HCN Channel Surface Expression by a Novel C-Terminal Protein-Protein Interaction
J. Neurosci. Santoro et al.
24: 10750
Supplemental data
Files in this Data Supplement:
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Supplemental Figure 1. Dendritic targeting of TrkB protein is not altered in HCN1-/- or HCN1f/f,cre+ mice. A-C anti-TrkB antibody staining by immunofluorescence in the neocortex of a wild-type (A), HCN1f/f,cre (B), or HCN1-/- (C) mouse. Imaging was performed by confocal microscopy (10x objective); all images represent a single focal plane, and were collected at the point of maximum labeling intensity for each section, using identical conditions for all samples. D,E Confocal images taken at higher magnification (40x objective) show detail of apical dendrite labeling in the neocortex of a wild-type (D) or HCN1-/- (E) mouse.
Materials and Methods. As described for HCN1 and TRIP8b protein distribution analysis (see main text), immunohistochemistry was carried out on free-floating sections (40 mM) from adult mouse brain (age 8-12 weeks), cut after perfusion with 4% PFA/PBS. Slices were washed in PBS, blocked with PBS + 0.1% Triton + 3% goat serum, and primary antibody labeling was carried out overnight at 4ºC in blocking solution. The affinity purified rabbit polyclonal antibody against the extracellular portion of TrkB (TrkB23-36) was a generous gift of Stu Feinstein (University of California Santa Barbara, CA) and was used at a 1:100 dilution (2 mg/ml). Secondary antibody labeling was performed for 2-4 hs at room temperature in blocking solution, using rhodamine-red-X-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, dilution 1:200). Labeling was detected using confocal microscopy, as described in figure legend.
