TUNEL assay was performed on tibial growth plate sections of three-week old mice using a fluorometric TUNEL kit (Promega, G3250) according to manufacturer's protocol as describe previously [49 ].The slides were mounted with Vectashield with DAPI (Vector Laboratories, H1200) and imaged using the CoolSNAP ES Olympus BX51 camera and associated Metaview software. In order to obtain a reliable estimate of number of apoptotic hypertrophic cells in the growth plate, the green fluorescent apoptotic cells within the HZ were counted in three slides, at least 50 μm apart, per mouse and five animals for each genotype. The total number of hypertrophic chondrocytes within the HZ was defined by the number of DAPI-stained nuclei (as determined using ImageJ software). The apoptosis rate was defined as a percentage of the total number of hypertrophic cells within HZ. All data were analysed by one-way ANOVA for statistical significance.
H 1200
Manufactured by Olympus
The Olympus H-1200 is a state-of-the-art laboratory equipment designed for high-performance microscopy applications. It features advanced optical capabilities and a robust construction to ensure precise and reliable performance.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti γh2ax, by Merck Group (2 mentions)
Rabbit igg f ab 2 fragment cy3, by Merck Group (2 mentions)
Fluorometric tunel kit, by Promega (1 mentions)
Coolsnap es bx51 camera, by Olympus (1 mentions)
Cm3050, by Leica (1 mentions)
Cellsens standard imaging software, by Olympus (1 mentions)
Cellular senescence plate assay kit spider βgal, by Dojindo Laboratories (1 mentions)
Cell count normalization kit, by Dojindo Laboratories (1 mentions)
X gal solution, by Merck Group (1 mentions)
G9023, by Merck Group (1 mentions)
Nb110 40763, by Novus Biologicals (1 mentions)
Rabbit anti 53bp1, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg fitc antibody, by Merck Group (1 mentions)
5 protocols using h 1200
1
Quantifying Apoptosis in Growth Plate
2
Immunofluorescence Staining of DNA Damage Markers
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Cells grown on cover glasses were fixed with 4% paraformaldehyde (PFA), then used 0.3% Triton X-100 to permeabilize. Or in some experiments cells were fixed and permeabilized with methanol:ethanoic acid (3:1) for 15 min. Cells were blocked for 30 min at 37 °C with 3% BSA in PBS supplemented with 0.2% Triton X-100. Blocked cells were then incubated for 40 min in 37 °C with diluted primary antibodies: Mouse anti-γH2AX (Millipore, 05–636), Mouse anti-ATM-pS1981 (Cell Signaling, 4526S), γH2AX Rabbit anti-53BP1 (Cell Signaling, 3428P), Rabbit anti-BRCA1-pS1524 (Bethyl, A300-001A), Rabbit anti-RAD51 (Santa, sc-8349). After washed by PBS, cover glasses were incubated for 40 min in 37 °C with secondary antibodies: Rabbit IgG F(ab′)2 fragment-Cy3 (Sigma) and goat anti-Mouse IgG-FITC antibody (Santa). After mounting with DAPI (VECTOR, H-1200), pictures were captured using an Olympus fluorescence microscope (BX 51) and analyzed with Image-Pro Plus. 100–200 cells were counted for quantitative immunofluorescence assay from three independent experiments.
For histological staining, freshly collected cervical tissues were embedded with optimal cutting temperature compound (O.C.T, Sakuraus, 4583) after using PBS clean, immediately frozen with liquid nitrogen and stored in -80 °C. Frozen tissue blocks were sectioned at 5 microns using microtome (LEICA, CM3050).
For histological staining, freshly collected cervical tissues were embedded with optimal cutting temperature compound (O.C.T, Sakuraus, 4583) after using PBS clean, immediately frozen with liquid nitrogen and stored in -80 °C. Frozen tissue blocks were sectioned at 5 microns using microtome (LEICA, CM3050).
3
Senescence-Associated β-Galactosidase Staining Assay
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Transfer the micro cover glass with the fixed cells from step 4.a.iii. into 24-well plate filled with PBS.
Remove the PBS.
Add 1 mL of Staining mixture including X-gal Solution (Sigma, CS00030).
Seal the plate with Parafilm.
Incubate the cells at 37°C for 16–20 h without CO2.
Wash the cells three times with 1 mL of PBS.
Cover with Mounting Medium with DAPI (VECTASHIELD, H-1200) and observe the cells on DIC and DAPI images with Olympus cellSens Standard Imaging Software using microscope (100× magnification, BX53, Olympus). Count more than 200 DAPI-positive cells manually, and calculate the percentage of SA-β-gal-positive cells per condition in each experiment. Please refer to (Yamamoto-Imoto et al., 2022 (link)).
4
Immunohistochemical Detection of TRPA1
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The cells were seeded into the wells of chambered slides, washed with PBS, fixed with ice cold methanol (10 min), and blocked with 5% normal goat serum in PBS (1 h at RT). A primary antibody recognizing the N-terminus of the human TRPA1 protein (NB110-40763, Novus Biologicals) was diluted in 1% normal goat serum in PBS (1:200) (G9023, Sigma) and applied 1h under agitation at RT. Cells without primary antibody were used as immunospecificity control. After washing with PBS (3 × 5 min), a secondary antibody that was diluted in 1% normal goat serum (1:200) (Cy2 anti-rabbit IgG, 111-225-144, Jackson Immuno Research) was applied for 1 h at RT under agitation. Next, cells were washed with PBS (3 × 5 min), coverslipped with 1–2 drops of antifade mounting medium with DAPI (VECTASHIELD; H-1200), and imaged with a fluorescence microscope (Olympus IX51).
5
Immunofluorescent Assay for DNA Damage
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Cells grown on cover slips were fixed with 4% paraformaldehyde (PFA), and permeabilized in 0.3% Triton X-100. Cells were blocked at 37 °C for 30 min with 3% BSA in PBS supplemented with 3% donkey serum and 0.2% Triton X-100. Fixed cells were then incubated with diluted primary antibodies: Rabbit anti-53BP1 (Cell Signaling, 3428P), Mouse anti-γH2AX (Millipore, 05–636), Rabbit anti-NBS1-pSerine 343 (Epitomics, 2194–1-1), BRCA1-pS1524 (Bethyl, A300-001A), Mouse anti-TP53-pSerine 15 (Cell Signaling, 2524); Rabbit anti-RPA32-pSerine 33 (NOVUS, NB100–544), Rabbit anti-ssDNA (IBL,18731). After extensive wash by PBS, coverslips were incubated with secondary antibodies (Rabbit IgG F(ab′)2 fragment-Cy3 and goat anti-Mouse IgG-FITC antibody, Sigma) for 30 min. After mounting with DAPI solution (VECTOR, H-1200), images were obtained using an Olympus epifluorescent microscope (BX 51) and analyzed with Image-pro plus (Applied Imaging). 100–200 cells or MN from 3 independent experiments were counted for quantitative immunofluorescent assays.
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