For the detection of oligonucleosomal DNA cleavage via terminal deoxynucleotidyl transferase end labeling (TUNEL), tissue sections were deparaffinized in toluene (2 × 5 min), rehydrated in distilled water (5 min), and pretreated with 3% citric acid (60 min) to remove tissue calcification. Endogenous peroxidase was quenched by incubating sections for 15 min in 0.9% hydrogen peroxide. Thereafter, TUNEL was performed using an ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Merck-Millipore, Burlington, MA, USA) according to the instructions of the manufacturer. Slides were observed with an Olympus BX61 microscope at 20× magnification, and pictures were taken with the Olympus Stream Essentials 1.9 software. Quantification of the TUNEL staining was performed with the “color threshold” function of ImageJ, using the color space RGB and the same parameters for each analyzed picture.
Stream essentials 1
Manufactured by Olympus
Stream Essentials 1.9 software is a core imaging application designed to control and acquire data from Olympus microscopes and cameras. It provides a user-friendly interface to capture, analyze, and share microscopy images and videos.
Automatically generated - may contain errors
2 protocols using stream essentials 1
1
Detection of Apoptotic DNA Fragmentation
2
Immunohistochemical Staining Protocol
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After 60 min incubation in Target Retrieval Solution, High pH (Dako, Agilent), at 98°C in a PT Link machine (Dako), the sections were washed twice with Envision FLEX Washing buffer, followed by incubation with peroxidase blocking reagent for 5 min. After 2 new wash steps, the sections were incubated for 30 min in the primary antibody solution (1:100), followed by 30 min in the secondary antibody solution (EnVision FLEX/HRP). Finally, the sections were incubated for 30 min in diaminobenzidine solution. The counterstaining (nuclei) is done automatically by the Leica ST5010 Autostainer XL. Slides were observed with an Olympus BX61 microscope at 20x magnification and pictures were taken with the Olympus Stream Essentials 1.9 software.
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