After 4 h of cooling at 10 °C and an additional 24 h of rewarming at 37 °C, neurons cultured on glass coverslips were incubated in the presence of 10 µg/ml propidium iodide (PI) (Cayman Chemical, Ann Arbor, USA) diluted in phosphate-buffered saline (PBS), and co-stained with 1 µg/ml Hoechst 33342 (Life Technologies) for 25 min at 37 °C. Cells were then fixed in ice-cold buffered formaldehyde (4%) for 15 min, washed twice in PBS, and placed in a histology mounting medium (Sigma-Aldrich) on a glass slide. The prepared material was imaged using a fluorescence microscope (Leica DMI 4000B, Germany) and LAS X SP8 software. Counting of total cells (blue nuclei) and necrotic cells (red-PI positive and round) was performed on two to three images from three coverslips as replicates. Collected data were statistically analyzed using Prism software (version 6.01 for Windows, La Jolla, CA).
Histology mounting medium
Manufactured by Merck Group
Histology mounting medium is a clear, colorless, and viscous liquid used in the preparation of microscope slides for histological analysis. It serves the core function of securely adhering and preserving thin tissue sections or cells onto the slide surface, enabling their examination under a microscope.
Automatically generated - may contain errors
2 protocols using histology mounting medium
1
Quantifying Neuronal Necrosis Using PI/Hoechst Staining
2
Detection of Apoptosis in Carotid Arteries
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Apoptosis of VSMCs in ligated carotid arteries was detected using a TUNEL Andy Fluor™ 488 Apoptosis Detection Kit (GeneCopoeia A050) according to manufacturer's instructions. Briefly, sections were deparaffinized and rehydrated, permeabilized by Proteinase K solution, incubated with TdT reaction cocktail, and labeled with Andy Fluor™ 488-Streptavidin staining solution. Sections were mounted with histology mounting medium (Sigma) supplemented with 40,6-diamidino-2-phenylin-dole (DAPI; H-1200, VECTASHIELD) for counterstaining DNA. Sections incubated with TdT reaction cocktail without terminal transferase were used as negative controls. Images were taken by a confocal microscope (DMI 4000B; Leica Microsystems, Wet-zlar, German) and quantitated by Image J software as described previously [27] (link).
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