Cells were fixed in 4% paraformaldehyde solution for 15 min at room temperature (25°C). Cells were permeabilized with 0.1% Triton X-100 solution for 10 min. Cells were washed with PBS on 3 times and incubated with 5% BSA blocking buffer for 30 min at room temperature (25°C). Then incubated with primary antibodies against LC3, LAMTOR1 and AMPK at 4°C overnight. Remove the primary antibodies and cells were washed with PBS on 3 times. Next, cells were incubated with secondary antibodies to Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (H+L) and Cy3-labeled Goat Anti-Mouse IgG (H+L) (Beyotime Biotech. Inc.; Shanghai, China) to avoid the light for 2 h at room temperature (25°C). Cells were washed with PBS on 3 times. The nuclei were stained using diamidino-2-phenylindole (DAPI ) solution for 10 min at room temperature (25°C). Images were captured using a TCS SP8 STED high-resolution laser confocal microscope (Leica; Wetzlar, Germany).
Tcs sp8 sted high resolution laser confocal microscope
Manufactured by Leica
Sourced in Germany
The Leica TCS SP8 STED is a high-resolution laser confocal microscope. It is designed to provide researchers with advanced imaging capabilities, including super-resolution techniques such as STED (Stimulated Emission Depletion) microscopy. The core function of the TCS SP8 STED is to enable high-resolution imaging and analysis of biological samples.
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Lab products found in correlation
4 protocols using tcs sp8 sted high resolution laser confocal microscope
1
Immunofluorescent Staining of Autophagy Markers
2
AMPK Regulation of Autophagy in Osteoclasts
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Osteoclasts were transfected with EGFP‐pmCherry‐LC3 plasmid (500 ng/well) for 12 hours and subjected to different treatments. The cells were fixed and treated with anti‐quenching agent in cell culture slides. LC3 puncta (EGFP‐LC3 represents autophagosomes and pmCherry‐LC3 represents autolysosomes) were identified using a TCS SP8 STED high‐resolution laser confocal microscope (Leica).
The siRNA targeting sequences (5′‐3′) are as follows: AMPKα1/2, sense: GCUAUCUUCUGGACUUCAA, antisense: UUGAAGUCCAGAAGAUAGC. As a control siRNA, we used a missense siRNA. Cells were transfected with siRNAs using Lipofectamine™ 3000 Transfection Reagent. Cell culture medium was then replaced with fresh α‐MEM medium incubated for 3 days. The medium was replaced every 2 days. At the end of incubation, various treatments were applied.
The siRNA targeting sequences (5′‐3′) are as follows: AMPKα1/2, sense: GCUAUCUUCUGGACUUCAA, antisense: UUGAAGUCCAGAAGAUAGC. As a control siRNA, we used a missense siRNA. Cells were transfected with siRNAs using Lipofectamine™ 3000 Transfection Reagent. Cell culture medium was then replaced with fresh α‐MEM medium incubated for 3 days. The medium was replaced every 2 days. At the end of incubation, various treatments were applied.
3
Visualizing Autophagy and Osteoclastogenesis
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EGFP‐pmCherry‐LC3 plasmid (500 ng/well) was transfected into cells using JetPRIME® transfection reagent for 24 hours. Cells were fixed in 4% paraformaldehyde solution for 15 minutes at room temperature. LC3 puncta were captured by the TCS SP8 STED high‐resolution laser confocal microscope (Leica, Germany).
Beclin1 siRNA plasmid sequences (sense 5′‐3′: GUACCGACUUGUUCCCUAUtt, antisense 5′‐3′: AUAGGGAACAAGUCGGUACtt), EGFP (pcDNA3.1‐EGFP) and c‐Fos (pcDNA3.1‐c‐fos) were transfected into cells using JetPRIME® transfection reagent for 24 hours. All medium was replaced by α‐MEM supplement with M‐CSF (30 ng/mL) and RANKL (60 ng/mL), and added the OPG (40 ng/mL) or not.
Beclin1 siRNA plasmid sequences (sense 5′‐3′: GUACCGACUUGUUCCCUAUtt, antisense 5′‐3′: AUAGGGAACAAGUCGGUACtt), EGFP (pcDNA3.1‐EGFP) and c‐Fos (pcDNA3.1‐c‐fos) were transfected into cells using JetPRIME® transfection reagent for 24 hours. All medium was replaced by α‐MEM supplement with M‐CSF (30 ng/mL) and RANKL (60 ng/mL), and added the OPG (40 ng/mL) or not.
4
Immunofluorescence Staining of Runx2 in Cells
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The cultural medium was discarded and washed with PBS twice. Cells were fixed in 4% paraformaldehyde solution for 30 min at room temperature (25 °C). The PBS was discarded and cells were mixed with 0.4% Triton X-100 solution for 10 min. The transmembrane solution was removed and combined with 5% BSA blocking buffer for 30 min at room temperature (25 °C). Cells were incubated with primary antibodies to Runx2 and IgG for 12 h, the latter serving as a negative control. We re-collected the primary antibodies and washed them with PBS 5 times. Then, the second immunofluorescence antibody was incubated for 2 h in the dark. Phalloidin staining was added into the cells for 10 min at room temperature (25 °C). Cells were washed with PBS 5 times and we added the Hoechst 33258 staining for 10 min at room temperature (25 °C). The images were recorded using a TCS SP8 STED high-resolution laser confocal microscope (Leica, Germany).
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