Hyperfilm β max

In situ hybridization histochemistry was performed on a separate cohort consisting of CES and control rats that were sacrificed on P19. The ISH method has been described in detail previously (Avishai-Eliner et al., 2001 (link), Ivy et al., 2008 (link)). Briefly, 20 μm coronal sections were collected on gelatin-coated slides and stored at −80 °C. Sections were thawed, air dried, fixed in paraformaldehyde, dehydrated, and rehydrated through graded ethanols, then exposed to 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8) and dehydrated. Prehybridization and hybridization steps were performed at 40 °C in a humidified chamber. Following one hour of prehybridization, sections were hybridized overnight (20 h) with a deoxyoligonucleotide probe complementary to the coding region of CRH mRNA and 3′-end-labeled with ³⁵S-dATP. Sections were then washed and apposed to film (Hyperfilm β-Max; Amersham, Arlington Heights, IL) for 7–14 days.

For analysis and quantification of the lipid composition of cells and exosomes secreted from the cells, we first performed [¹⁴C]-acetate metabolic labeling of the cells during 72h. After that, cells were stimulated as indicated above for exosome secretion, cells separated from supernatants by centrifugation, and exosomes isolated as indicated above. After that, cell or exosome lipids were extracted with chloroform/methanol (2:1, v/v) and lipids separated by thin-layer chromatography (TLC). Phospholipid separation was made using chloroform/methanol/32% ammonia (65:35:5, v/v; [68 (link)]) as eluent. Neutral lipid separation was made using the standard eluent hexane/ethyl ether/acetic acid (70:30:1, v/v; [69 (link)]). Radiolabelled lipids in TLC plates were located by autoradiography (Hyperfilm β-max, Amersham) at room temperature for 2 days and radioactivity quantified by liquid scintillation counting of scrapped silicagel. In all TLC analysis, positions of the authentic lipids were identified by running commercial standards of all lipid and phospholipid classes (Sigma, Madrid, Spain) in the same plates, and revealing them with iodine.