Blastocyst-stage embryos were isolated by flushing uteri with M2 Medium (Sigma). Embryos were collected between E3.5 and E4.0, washed with M2 Medium, fixed in 2% paraformaldehyde, and permeablized with 0.25%Triton-X/PBS. Embryos were blocked with 2.5% donkey serum/0.5%PBS-TSP/PBS, and incubated with primary antibodies (EGFL7, CDX2) overnight at 37°C in block. Blastocysts were incubated with secondary antibodies and mounted with Prolong Gold+DAPI using Fastwell spacers (Sigma-Aldrich).
Prolong gold dapi
Manufactured by Merck Group
Prolong Gold DAPI is a fluorescent stain used in molecular biology and cell biology applications to label and visualize DNA. It is a sensitive and specific stain that binds to the minor groove of double-stranded DNA. The stain exhibits blue fluorescence when bound to DNA and can be detected using standard fluorescence microscopy techniques.
Automatically generated - may contain errors
3 protocols using prolong gold dapi
1
Blastocyst Immunostaining for EGFL7 and CDX2
2
H2AX Foci Quantification in HMVEC-L
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H2AX immunostaining was performed as described [23] . Briefly, HMVEC-L were seeded at 5000 cells/cm² on glass coverslips coated with 1% gelatin and grown to confluence. One hour, 7
days and 14 days post-15 Gy, cells were fixed in 4% PFA, and permeabilized with 0.1% Triton X-100. Cells were incubated with γH2AX antibody (9718, Cell Signaling) O/N at 4°C, then with anti-IgG-Alexa568 (A11036, Invitrogen) 1 h, at RT and counterstained with Prolong Gold DAPI (Sigma). H2AX foci number in nucleus section were observed under confocal microscope (Nikon A1 630x magnification) and count was processed using ImageJ (NIH). Number of foci per cell was reported on the total cell number per field representing a mean of 350 cells from 9 fields (3 independent experiments).
days and 14 days post-15 Gy, cells were fixed in 4% PFA, and permeabilized with 0.1% Triton X-100. Cells were incubated with γH2AX antibody (9718, Cell Signaling) O/N at 4°C, then with anti-IgG-Alexa568 (A11036, Invitrogen) 1 h, at RT and counterstained with Prolong Gold DAPI (Sigma). H2AX foci number in nucleus section were observed under confocal microscope (Nikon A1 630x magnification) and count was processed using ImageJ (NIH). Number of foci per cell was reported on the total cell number per field representing a mean of 350 cells from 9 fields (3 independent experiments).
3
Mitochondrial Morphology and Mass Analysis
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HMVEC-L were incubated with Mitotracker™ Red CMXRos (Invitrogen) for 30 min at 37°C before 4% PFA fixation. Mitochondria morphology was examined on slides counterstained with Prolong Gold DAPI (Sigma) under confocal microscope (Nikon A1 630x magnification).
Mitochondrial mass per cell were assessed on 15000 cells by FacsCalibur and CellQuest analysis.
Mitochondrial mass per cell were assessed on 15000 cells by FacsCalibur and CellQuest analysis.
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