Immunofluorescence analysis of HCN2 was performed using 70% confluent log phase Neuro-2A cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with HCN2 Rabbit Polyclonal Antibody (PA1-918) at 2ug/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membranous localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Mouse, Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic Peptide: G(874) L D P Q D S A R S R L S S N L(889)|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Western Blot (WB)||1:500|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
PA1-918 detects the HCN2 from rat samples.
PA1-918 has been successfully used in Western blot procedures. By Western blot, this antibody detects bands at ~100 kDa and ~115 kDa representing the non-glycosylated and glycosylated form of HCN2 in rat brain extract.
The PA1-918 immunizing peptide corresponds to amino acid residues 874-889 from human HCN2.
Hyperpolarization-activated cation channels of the HCN gene family such as HCN2, contribute to spontaneous rhythmic activity in both heart and brain. HCN2 is a member of a family of pacemaker channels activated by hyperpolarization and regulated by cyclic nucleotides. HCN1 and HCN2 play an important role for motor learning and neuronal integration by cerebellar Purkinje cells; as well as, shaping autonomous activity of single neurons and the periodicity of network oscillations.
The HCN1 expression is highly enriched in cerebral cortex, hippocampus, cerebellum, and facial motor nucleus. HCN2 is highly abundant in mamillary bodies, pontine nucleus, ventral cochlear nucleus, and nucleus of the trapezoid body. These variations in regional specificity of HCN channels could generate important differences in neuronal pacemaker activity across brain systems.
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