Chicken anti vimentin

The desired brain sections containing the SFO and OVLT were initially washed free‐floating in PBS. Next, brain tissue was permeabilized with 0.1% Triton X100 (Sigma) for 20 minutes at room temperature (RT), following further washing in PBS. Slices were then blocked in 5% normal donkey serum (NDS; Sigma) and 0.05% Triton X100 at RT for 30 minutes. A 48 hours incubation at 4°C in a PBS solution containing 0.05% Triton X100, 0.5% NDS and the primary antibodies rabbit anti‐GFP (1:1000, Abcam), and chicken anti‐vimentin (1:4000, Abcam). Following this incubation, slices were washed in PBS and transferred to PBS solution of secondary antibodies for 24 hours at 4°C; donkey anti‐rabbit Cy5 (1:800, Jackson ImmunoResearch) and donkey anti‐chicken Alexa 488 (1:800, Abcam). The secondary antibodies were then washed off in PBS and the slices were mounted on gelatine‐coated glass slides using VectaShield medium (Vector Laboratories, USA). Slices were then imaged on a confocal microscope (Leica SP5 upright) under the 20x objective. Z‐stacks were acquired in 3 µm steps and viewed as maximal intensity projections.

Hearts were flash frozen in liquid nitrogen cooled isopentane and cryosectioned at 10 μm. Phalloidin (Alexa Fluor 594, Life Technologies, cat. A12381) and DAPI (Life Technologies, cat. D1306) stained sections were used to identify the scars. Sections were fixed in 100% acetone at −20 °C for 5 min then blocked for one hour in blocking solution containing (in mM) 20 Tris pH 7.4, 155 NaCl, 2 EGTA, 2 MgCl₂ and 5% v/v Donkey Serum (Jackson Immunoresearch). Primary antibodies were incubated overnight at 4 °C or one hour at room temperature. Primary antibodies were diluted in blocking buffer as follows: Mouse anti-beta galactosidase (Promega, cat. Z3781) 1:100, chicken anti-Vimentin (Abcam, cat. 24525), rabbit anti-Cx43 (Millipore, cat. AB1727). Alexa conjugated secondary antibodies (Jackson Immunoresearch Laboratories) were used in a 1:200 dilution.