In brief, the section was incubated with primary antibodies against Caspase8 (1:100, 13423‐1‐AP; Proteintech) GSK3β (1:100, 22104‐1‐AP; Proteintech) and Phospho‐GSK3β (Ser9) (1:100, 67558‐1‐Ig; Proteintech) at 4°C overnight, followed by incubation with secondary antibodies including Alexa Fluor 488 Conjugate (1:100; #ZF‐0512; ZSGB‐BIO) and Alexa Fluor 594 Conjugate (1:100; #ZF‐0513) at room temperature for 2 h. The images were investigated under a fluorescence microscopy (Olympus). The expression of target proteins was quantified according to integrated optical density (IOD) value with ImageJ software.
Alexa fluor 488 conjugate
Manufactured by ZSGB-BIO
Sourced in China
Alexa Fluor® 488 Conjugate is a fluorescent dye used in various biological applications. It has an excitation maximum at 495 nm and an emission maximum at 519 nm, making it suitable for detection and visualization techniques that utilize green fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 conjugate, by ZSGB-BIO (6 mentions)
Sc 515 930, by Santa Cruz Biotechnology (3 mentions)
Ab190114, by Abcam (2 mentions)
Confocal microscope, by Olympus (2 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
Ab101465, by Abcam (1 mentions)
Ab81289, by Abcam (1 mentions)
Lin28a, by Santa Cruz Biotechnology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Ab231303, by Abcam (1 mentions)
Zli 9557, by ZSGB-BIO (1 mentions)
Sc 73408, by Santa Cruz Biotechnology (1 mentions)
8 protocols using alexa fluor 488 conjugate
1
Quantitative Analysis of Caspase8, GSK3β, and Phospho-GSK3β
2
Immunofluorescence Analysis of Mitochondrial Proteins
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The transfected cells were fixed in 4% paraformaldehyde lasting 30 min. And, 0.5% Triton X-100 was applied to cover the slides and incubated at room temperature for 10 min. The sample was blocked with 10% goat serum for 1 h, and then washed thrice with 1% serum blocking solution. The primary antibody against MCU (1:100; sc-515,930; Santa Cruz, USA), MICU1 (1:1000; ab190114; Abcam, USA) and MICU2 (1:1000; ab101465; Abcam, USA) was added dropwise to the slices. After washing six times with 1% serum blocking solution, the sections were incubated with secondary antibodies Alexa Fluor® 488 Conjugate (1:100; ZF-0512; ZSGB-BIO, China) and Alexa Fluor® 594 Conjugate (1:100; ZF-0513; ZSGB-BIO, China) at room temperature for 2 h. Then, the sections were incubated by DAPI at room temperature for 5 min in the dark. The images were captured using an immunofluorescence microscope. The Image-Pro Plus 6.0 image processing system was used to measure the average optical density of the protein.
3
Immunofluorescence analysis of MCU and CD34
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Paraffin-embedded tissue sections were incubated with primary antibodies labeled with fluorescent substance including anti-MCU (1:100; sc-515,930; Santa Cruz, USA) and anti-CD34 (1:100; ab81289; Abcam, USA) overnight at 4 °C. The sections were then incubated with Alexa Fluor® 488 Conjugate (ZSGB-BIO, #ZF-0512, 1:100, China) and Alexa Fluor® 594 Conjugate (ZSGB-BIO, #ZF-0513, 1:100, China) secondary antibodies at room temperature for 2 h. The results were investigated under an immunofluorescence microscope.
4
Immunofluorescence Staining of piPSCs
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After twice washing with PBS, the piPSCs (cultured for 5 days) were fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 15 min. We used 0.1% Triton-100 to perforate the membranes at room temperature for 10 min. Then, 10% FBS was used to block the membranes at room temperature for 1 h. The membranes were incubated with primary antibodies, including LIN28A (1∶200; Santa Cruz Biotechnology, USA) and FLAG (1∶1 000; Sigma-Aldrich, USA), for 12 h at 4 °C and then washed three times with PBS. The membranes were then incubated with goat anti-mouse IgG (H+L) secondary antibody Alexa Fluor 488 conjugate (1∶500; ZSGB-BIO, China) at room temperature for 1 h and nuclei were stained with Hoechst33342 (1∶1 000) at room temperature for 5 min (Ma et al., 2019 (link); Wei et al., 2021 (link)).
5
Immunofluorescence Analysis of Keap1, Nrf2, and HO-1
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Following deparaffinization and hydration, specimens were cut into 4 μm thickness. The sections were incubated with fluorescent substance-labeled primary antibodies against Keap1 (1 : 200; Proteintech; China; 60027-1-Ig), Nrf2 (1 : 100; Proteintech; China; 16396-1-AP), and HO-1 (1 : 100; Proteintech; China; 27282-1-AP) overnight at 4°C. Then, sections were incubated with secondary antibodies including Alexa Fluor® 488 Conjugate (1 : 100; #ZF-0512, ZSGB-BIO, China) and Alexa Fluor® 594 Conjugate (1 : 100, #ZF-0513, ZSGB-BIO, China) at room temperature for 2 h. The images were observed under a confocal microscope (Olympus, Japan).
6
Immunofluorescence Profiling of EMT Markers
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Tissue sections were incubated with primary antibodies labeled with fluorescent substance against ADAR1 (1:50, sc-73408, SANTA CRUZ BIOTECHNOLOGY, USA ), CALR (1:100; 27298-1-AP; Proteintech, China), β-catenin (1:100; 51067-2-AP; Proteintech, China), E-cadherin (1:150; ab231303; Abcam, USA) and Vimentin (1:1000; 10366-1-AP; Proteintech, China) overnight at 4 °C, followed by incubation with secondary antibodies Alexa Fluor® 488 Conjugate (1:100; #ZF-0512; ZSGB-BIO) as well as Alexa Fluor® 594 Conjugate (1:100; #ZF-0513; ZSGB-BIO) at room temperature lasting 2 h. The nuclear was counterstained by DAPI (ZLI-9557; ZSGB-BIO, China) for 5 min. The images were captured under a fluorescence microscope (BX61, Olympus, Japan).
7
Mitochondrial Protein Localization Visualization
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Paraffin-embedded tissue sections were incubated with primary antibodies labeled with fluorescent substance against MCU (1 : 100; sc-515930; Santa Cruz, US), MICU1 (1 : 1000; Abcam, USA; ab190114), MICU2 (1 : 1000; Abcam, USA; ab101465), and Nrf2 (1 : 1000; Proteintech, China; 16396-1-AP) overnight at 4°C, followed by secondary antibodies Alexa Fluor® 488 conjugate (1/100; ZSGB-BIO, China; ZF-0311, 1/100; ZSGB-BIO, China; ZF-0514) and Alexa Fluor® 594 conjugate (#ZF-0513, 1 : 100, ZSGB-BIO, China) at room temperature for 2 h. The images were observed under an immunofluorescence microscope.
8
Immunofluorescence Analysis of Keap1, Nrf2, and HO-1
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CAL-27 cells were seeded in a 24-well plate (3 × 103 cells/well). Following fixing cells for 15 min, the cell-climbing slide was washed using 0.5% Triton× 100 PBS solution. Then, sections were blocked with 3% BSA and incubated with primary antibodies against Keap1 (1 : 200; Proteintech; China; 60027-1-Ig), Nrf2 (1 : 100; Proteintech; China; 16396-1-AP), and HO-1 (1 : 100; Proteintech; China; 27282-1-AP) overnight at 4°C, followed by incubation with secondary antibodies including Alexa Fluor® 488 Conjugate (1 : 100; #ZF-0512, ZSGB-BIO, China) and Alexa Fluor® 594 Conjugate (1 : 100, #ZF-0513, ZSGB-BIO, China) at room temperature for 2 h. The images were investigated under a confocal microscope (Olympus, Japan).
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