Embryos were fixed in 4% PFA in PBS and infiltrated with 30% sucrose in PBS before being mounted in O.C.T. compound (Sakura Finetek United States Inc., Torrance, CA, United States). Sections (8 μm) were deposited on glass slides. Apoptotic cells were identified using the In Situ Cell Death Detection Kit, Fluorescein (Sigma-Aldrich Corp.) according to the manufacturer’s instructions for the treatment of cryopreserved tissue sections. Sections were mounted in VECTASHIELD® Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, United States) and photographed using an Axiocam 506 mono digital camera (Carl Zeiss, Inc.) fitted onto an Axio Observed 7 fluorescence microscope (Carl Zeiss, Inc.). All positive signals were confirmed by DAPI staining. The percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells was determined in three embryos per genotype per timepoint, with up to four sections analyzed per individual embryo. Analyzed sections within a given embryo were 5–10 sections apart, representing a distance of 40–80 μm. Graphed data represent averages from three independent embryos per timepoint. Statistical analyses were performed on values from individual sections with Prism 8 (GraphPad Software, Inc.) using a two-tailed, unpaired t-test with Welch’s correction and Welch and Brown-Forsythe ANOVA tests.
Axio observed 7 fluorescence microscope
Manufactured by Zeiss
Sourced in United States
The Axio Observed 7 is a fluorescence microscope designed for laboratory applications. It provides high-resolution imaging capabilities for a wide range of sample types.
Automatically generated - may contain errors
2 protocols using axio observed 7 fluorescence microscope
1
TUNEL Analysis of Embryonic Apoptosis
2
Apoptosis Detection in Embryo Sections
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Embryos were fixed in 4% PFA in PBS and infiltrated with 30% sucrose in PBS before being mounted in O.C.T. compound (Sakura Finetek USA Inc., Torrance, CA, USA). Sections (8 µm) were deposited on glass slides. Apoptotic cells were identified using the In Situ Cell Death Detection Kit, Fluorescein (Sigma-Aldrich Corp.) according to the manufacturer's instructions for the treatment of cryopreserved tissue sections. Sections were mounted in VECTASHIELDÒ Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and photographed using an Axiocam 506 mono digital camera (Carl Zeiss, Inc.) fitted onto an Axio Observed 7 fluorescence microscope (Carl Zeiss, Inc.). All positive signals were confirmed by DAPI staining. The percentage of TUNEL-positive cells was determined in three embryos per genotype per timepoint. Statistical analyses were performed with Prism 8 (GraphPad Software, Inc.) using a two-tailed, unpaired t-test with Welch's correction and Welch and Brown-Forsythe ANOVA tests.
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