Alexa fluor 546 goat anti rabbit antibody

Coverslips containing purified ganglion cells were fixed in 4% paraformaldehyde in 0.01-M PBS [Cedarlane Laboratories, Burlington, ON; 5 min, room temperature (RT)]. RGCs were washed with PBS and treated with normal goat serum blocking solution (Sigma Aldrich, Oakville, ON; 20% in PBS, 30 min, RT). Coverslips were incubated for 18–24 h (4°C) with α₂δ₁ Ca²⁺ channel subunit primary antibody (affinity purified rabbit polyclonal, EPFPSAVTIKSWVDK, extracellular epitope; 1:200 in PBS; Alomone Laboratories, Jerusalem, Israel), then washed in PBS. Samples were incubated with Alexa Fluor 546 goat antirabbit antibody (Life Technologies Corp., Burlington, ON) for 2 h (RT), washed with PBS and mounted on microscope slides using VectaShield (Vector Laboratories, Burlington, ON). Cells were not permeabilized with Triton-X (omitted from all immunohistochemical buffers) to limit antibody staining to the cell surface. To determine the selectivity of the α₂δ₁ antibody, preadsorption control samples were performed in parallel using the supplied control peptide (Alomone Labs). The control peptide was incubated with the α₂δ₁ antibody (2:1 by weight, 4°C, overnight) prior to adding the suspension to the isolated RGCs. In addition, secondary antibody controls (omission of primary antibody) were performed to ensure selectivity of the Alexa Fluor 546 goat antirabbit for the α₂δ₁ antibody.

Fifteen minutes following a seizure, mice (including non-seizure controls) were transcardially perfused with 4% PFA/PBS, brains were harvested, and post-fixed for 2 h at RT (Houser, et al., 2008 (link)). Brains were cryoprotected in 30% sucrose/PBS and sectioned in the coronal plane at a thickness of 30 μm. Tissue slices were mounted on Superfrost Plus microscope slides (ThermoFisher Scientific) and were air-dried overnight at RT. Tissue sections were permeabilized with PBS-Tx and blocked for 1 hr at RT with 10% goat serum. pERK antibodies were diluted (1:500, Cell Signaling) in 1% goat serum in PBS-Tx and incubated overnight at 4°C. Tissue sections were rinsed thoroughly and incubated for 1 h at RT with Alexa Fluor 546 goat anti-rabbit antibody (1:500, Life Technologies), washed and mounted with Fluoromount-G antifade solution (Southern Biotechnology). Images were acquired using a Zeiss Z1 microscope equipped with an ORCA-Flash 4.0 digital camera (Hamamatsu) and Neurolucida software (MBF Bioscience).