Table 4.
Visualization of ion channel subunits in flat-mounted rat retina (Stradleigh et al., 2011)
| Antigen | HCN4 | |
| Primary Antibody | mouse monoclonal anti-HCN4 (clone N114/10, NeuroMab) | |
| Secondary Antibody | Alexa Fluor 488-conjugated donkey anti-mouse (#715-545-151, Jackson ImmunoResearch) | |
| Fixative | modified Davidson’s Fluid (mDF; Latendresse et al., 2002) | |
| Fixative Recipe (10 mL) | formalin (#F-1635, Sigma-Aldrich) | 3 mL |
| ethanol (100%) | 1.5 mL | |
| glacial acetic acid | 0.5 mL | |
| water | 5 mL | |
1
. Dissect whole retina in ice cold PBS, place vitreous side up on black nitrocellulose membrane filter (#HABG01300, Millipore), and flatten by applying suction.
2
. While applying suction, add 0.5 mL ice cold mDF dropwise to membrane-attached retina.
3
Immerse flat mount in 10 mL room temperature mDF, 30 min.
4
. Rinse and quench in Tris-buffered saline, 30 min at 4°C.
5
. Permeabilize in PBS (supplemented with 0.5% bovine serum albumin, 0.05% Triton X-100, 5% normal donkey serum), 18 hr at 4°C.
6
. Apply primary antibody in PBS, 18 hr at 4°C. Rin se in PBS.
7
. Apply secondary antibody in PBS, 18 hr at 4°C.
8
. Rinse in PBS and mount to slide.