Journal: Physiological Reports
Article Title: Positive chronotropic action of HCN channel antagonism in human collecting lymphatic vessels). Positive chronotropic action of HCN channel antagonism in human collecting lymphatic vessels
Figure Lengend Snippet: Human lymphatic vessels predominantly express HCN2. (a) RT‐PCR analysis of HCN2 (229 base pairs) and HCN3 (589 bp) amplified from thoracic duct (TD) and mesenteric lymphatic vessels (ML); HCN1 (597 bp) and HCN4 (232 bp) were consistently amplified from the control human RNA, dorsal root ganglia (DRG) and heart, respectively, while all lymphatic samples were negative. Samples are presented pairwise as reverse transcriptase positive (+) followed by reverse transcriptase negative (−). HCN2 immunoreactivity (red fluorescence, left panels) was observed in smooth muscle cells of (b) human thoracic duct and (c) mesenteric lymphatic vessels. (d) Antibody specificity was confirmed by the absence of staining when the antibody was preincubated with peptide control (green fluorescent nuclear stain). Scale bars denote 50 μm (top) and 100 μm (middle and bottom), * indicates lumen, and right panels present differential interference contrast (DiC) images of the same section.
Article Snippet: Thereafter, tissue was permeabilized with 0.25% Triton X‐100 in PBS for 10 min following by blocking with 2% bovine serum albumin (BSA) in PBS for 20 min. Tissue was incubated overnight in a dark, humidified chamber at 4°C with 1:50 rabbit polyclonal anti‐HCN2 (APC‐030‐AR; Alomone, Israel) preconjugated to ATTO‐594 fluorophore.
Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Fluorescence, Staining