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16

and had equal variance and mean, and was delivered to the preparation at 1000 Hz sample
rate in phase with the current clamp recordings. First order kernels were estimated using
the Lee-Shetzen cross-correlation approach, with the first order kernel equal to the weighted
cross-correlation between the stimulus and recorded response [80]. For the purposes of this
paper, we use the term impulse response to describe the first order kernel. This is because
photoreceptors can be approximated as linear-the power of the photoreceptor response is
comparable to the power of the first order kernel predicted response [79].

2.4 Immunohistochemistry and Western Blot

Immunohistochemical experiments were performed using a rabbit anti rat polyclonal HCN1
antibody (Sigma) and a rabbit anti human HCN3 antibody (Santa Cruz Biotech). Salaman-
der retina was embedded in low temperature gelling agar (Sigma) and cut into 40 μm sec-
tions with a vibratome. Sections were permeabilized and blocked with a 10% donkey serum
in phosphate buffered saline (PBS) with Triton overnight, and then incubated in primary
antibody in 3% donkey serum PBS for 5 days. The sections were washed and incubated
overnight in donkey-anti-goat antibodies tagged with Alexa-488 (Molecular Probes). They
were then mounted on slides and imaged on a Zeiss LSM 510 microscope.

Western blot experiments were performed using the same HCNl and HCN3 antibodies
as the immunohistochemistry. The anti-HCNl antibody’s epitope was residues 6-24 (near
the N terminus) of rat HCNl protein, representing the intracellular domain. A BLASTp
search in Pubmed demonstrated that this sequence is highly conserved among mouse, rat,



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