Optimal cutting temperature compound o c t

Hearts were fixed in 1% PFA overnight, placed in 20% sucrose, then incubated in a 1:1 mixture of 20% sucrose and Optimal Cutting Temperature compound (OCT; Sakura Finetek) overnight and frozen in OCT the next day. Whole aortic roots were sectioned at 10 µm with a Microm HM 505 E cryotome (Microm International) and adhered to Denville UltraClear Microscope Slides (Denville Scientific). Slides were frozen until further use. All analyses were performed using NIS-Elements software.

Necropsies were performed immediately subsequent to euthanasia at predetermined timepoints. Sample collection schemes were predetermined and standardized with minor variation among individual animals. A maximum of 28 anatomically distinct tissues were collected per animal. Tissue specimens were collected from the oral cavity, nasal cavity, soft palate, pharynx, larynx, trachea, lungs, lymph nodes and skin (Table 2) as previously described [35 (link)]. For each defined specimen, two 30 mg tissue samples were aliquoted into separate screw-cap 1.5 ml cryovials and frozen immediately in liquid nitrogen for transfer within 2 h to a -70°C freezer in which they were stored until the time of processing. An adjacent specimen from each tissue was placed in a cryomold, embedded in Optimal Cutting Temperature Compound (OCT) (Sakura Finetek, Torrance, CA), frozen on a bath of liquid nitrogen, and stored at—70°C for immunomicroscopy.

On GD 21, a day before parturition, animals—were deeply anesthetized with isoflurane in the anesthesia chamber and sacrificed by decapitation and fetuses were carefully separated. Two male fetuses from each dam were randomly selected from a similar position in the uterine horn. One pup per dam was used for immunoblotting while the other pup was used for immunohistochemistry. During normal development, male and female hippocampal development differ significantly and brain development in humans and animal is directly regulated by sex hormones (Yagi and Galea, 2019 (link); Mu et al., 2020 (link)). To accurately identify hippocampal-specific alterations induced by prenatal e-cig aerosol exposure without the confounding factor of sex, only male offspring were assessed in these experiments. Fetal whole brains from the male fetuses were extracted under a dissection microscope via craniotomy and serially washed in cold phosphate buffered saline (PBS). One of the fetal brains was dissected, meninges were removed, and hippocampi were micro-dissected in ice-cold HEPES buffer. Individual samples were then flash frozen and stored at −80°C until immunoblot analyses. For immunohistochemistry, the other whole fetal brain was embedded in optimal cutting temperature compound (OCT) (Sakura Finetek, Torrance, CA) and cryopreserved for immunofluorescence.