IF was performed as previously described (Leung et al. 2014 (link)). Briefly, U2OS cells expressing shGFP or shFKBP25 were directly seeded on coverslips for overnight incubation. For Etopside damage, cells were treated with 100 μmol/L Etopside for 20 mins, and then washed and incubated for 2 h. For the ionizing radiation experiments, cells were treated with 5 Gy delivered by a Faxitron X-Ray machine. After IR, cells were incubated for 2 h. After the indicated treatment, cells were pre-extracted with cytoskeletal (CSK) buffer for 5 min on ice, fixed with 2% (v/v) formalin for 15 min at room temperature, and blocked with PBS containing 3% bovine serum albumin (BSA). After blocking, the cells were incubated with primary antibody overnight. After 3× PBS washes, the cells were incubated with secondary antibody for 1 h at room temperature. Primary antibody used was RAD51 (ab133534; Abcam). The secondary antibody used was Alex Fluor 488 goat anti-mouse IgG (Invitrogen). After slide preparation, imaging was processed and analyzed with the Z-stacked setting using the FV10-ASW3.1 software on a Fluoview 1000 confocal microscope (Olympus).
Alex fluor 488 goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 goat anti-mouse IgG is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is designed to detect and bind to mouse immunoglobulin G (IgG) antibodies, allowing for fluorescent detection and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Rock1, by Abcam (3 mentions)
Fluoview 1000 confocal microscope, by Olympus (2 mentions)
Ab133534, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Access rt pcr system, by Promega (2 mentions)
Alex fluor 594 goat antirabbit igg, by Thermo Fisher Scientific (2 mentions)
Total rna isolation system, by Promega (2 mentions)
Mouse anti mf20, by Developmental Studies Hybridoma Bank (2 mentions)
Mouse anti ctnt, by Lab Vision (2 mentions)
Eclipse 80i, by Nikon (1 mentions)
Dapi nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Secondary anti mouse igg hrp antibody, by R&D Systems (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
Rabbit anti mlc2v, by Proteintech (1 mentions)
Mouse anti alpha actinin, by Merck Group (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
9 protocols using alex fluor 488 goat anti mouse igg
1
Immunofluorescence Staining of DNA Repair Foci
2
Oxidative Stress-Induced ROCK1 and p53 Assay
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The B3 cells were cultured in 24-well plates and treated with 200 µM H2O2 in media for 24 h. These cells were fixed with 4% paraformaldehyde for 10 min, permeabilized in 0.3% Triton X-100 for 10 min, blocked with 3% bovine serum albumin (BSA) for 30 min at 37 ℃. The primary antibodies of ROCK1 (1:200, Abcam) and p53 (1:200, Cell Signaling Technology) were first incubated at 37 ℃ for 1 h and then at 4 ℃ overnight. Alex Fluor 594 goat anti-rabbit.
IgG (Invitrogen, A11037) and Alex Fluor 488 goat anti-mouse IgG (Invitrogen, A11029) were as the secondary antibody incubated for 1 h at 37 ℃. Images were selected using a fluorescence microscope.
IgG (Invitrogen, A11037) and Alex Fluor 488 goat anti-mouse IgG (Invitrogen, A11029) were as the secondary antibody incubated for 1 h at 37 ℃. Images were selected using a fluorescence microscope.
3
Immunofluorescence Staining of DNA Repair Foci
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IF was performed as previously described (Leung et al. 2014 (link)). Briefly, U2OS cells expressing shGFP or shFKBP25 were directly seeded on coverslips for overnight incubation. For Etopside damage, cells were treated with 100 μmol/L Etopside for 20 mins, and then washed and incubated for 2 h. For the ionizing radiation experiments, cells were treated with 5 Gy delivered by a Faxitron X-Ray machine. After IR, cells were incubated for 2 h. After the indicated treatment, cells were pre-extracted with cytoskeletal (CSK) buffer for 5 min on ice, fixed with 2% (v/v) formalin for 15 min at room temperature, and blocked with PBS containing 3% bovine serum albumin (BSA). After blocking, the cells were incubated with primary antibody overnight. After 3× PBS washes, the cells were incubated with secondary antibody for 1 h at room temperature. Primary antibody used was RAD51 (ab133534; Abcam). The secondary antibody used was Alex Fluor 488 goat anti-mouse IgG (Invitrogen). After slide preparation, imaging was processed and analyzed with the Z-stacked setting using the FV10-ASW3.1 software on a Fluoview 1000 confocal microscope (Olympus).
4
Immunofluorescence Staining of Extracellular Matrix
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Example 5
Briefly, cells were fixed at 48 hours after seeding with 4% paraformaldehyde for 10 min and then permeabilized with 0.1% Triton X-100 for 5 min and followed by blocking with 5% bovine serum albumin (BSA) for 1 hour. The primary antibodies were mouse anti-collagen type I (1:400), or rabbit anti fibronectin (1:400) (CHEMICON) which incubated with the samples at 37° C. for 1 hour, and secondary antibodies were Alex Fluor 488 goat anti-mouse IgG (1:400), or chicken anti-rabbit IgG (1:400) (Invitrogen), and TRITC-conjugated Phalloidin (1:400) (CHEMICAON) which was applied at room temperature for 1 hour. 4′,6-diamidino-2-phenylindole (DAPI) (1:1000) (CHEMICON) was applied for 10 minutes to stain cell nuclei. Fluorescence images were captured using an upright fluorescent microscope (Eclipse 80i, Nikon).
5
Oxidative Stress Induces ROCK1 and p53 Expression
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The B3 cells were cultured in 24-well plates and treated with 200 µM H 2 O 2 in media for 24h. These cells were xed with 4% paraformaldehyde for 10 minutes, permeabilized in 0.3% Triton X-100 for 10 minutes, blocked with 3% bovine serum albumin (BSA) for 30 minutes at 37℃. The primary antibodies of ROCK1(1:200, Abcam) and p53 (1:200, Cell Signaling Technology) were rst incubated at 37℃ for 1h and then at 4℃ overnight. Alex Fluor 594 goat antirabbit IgG (Invitrogen, A11037) and Alex Fluor 488 goat anti-mouse IgG (Invitrogen, A11029) were as the secondary antibodyy incubated for 1h at 37℃. Images were selected using a uorescence microscope.
RNA extraction, reverse transcription, and quantitative RT-PCR Total cellular RNA was extracted from B3 cells using the Promega Total RNA Isolation System according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the Access RT-PCR System (Promega). The ROCK1 gene primer pairs for quantitative PCR assay were as follows: sense, 5′-TGTGACTGGTGGTCGGTT-3′ and antisense, 5-′GGTTTTTTGCTTCTTTT′G-3. Primer pairs for β-actin were as follows: sense, 5′-TCGTGCGTGACATTAAGGAG-3′, and antisense, 5′-ATGCCAGGGTACATGGTGGT′-3.
RNA extraction, reverse transcription, and quantitative RT-PCR Total cellular RNA was extracted from B3 cells using the Promega Total RNA Isolation System according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the Access RT-PCR System (Promega). The ROCK1 gene primer pairs for quantitative PCR assay were as follows: sense, 5′-TGTGACTGGTGGTCGGTT-3′ and antisense, 5-′GGTTTTTTGCTTCTTTT′G-3. Primer pairs for β-actin were as follows: sense, 5′-TCGTGCGTGACATTAAGGAG-3′, and antisense, 5′-ATGCCAGGGTACATGGTGGT′-3.
6
Oxidative Stress Induces ROCK1 and p53 Expression
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The B3 cells were cultured in 24-well plates and treated with 200 µM H 2 O 2 in media for 24h. These cells were xed with 4% paraformaldehyde for 10 minutes, permeabilized in 0.3% Triton X-100 for 10 minutes, blocked with 3% bovine serum albumin (BSA) for 30 minutes at 37℃. The primary antibodies of ROCK1(1:200, Abcam) and p53 (1:200, Cell Signaling Technology) were rst incubated at 37℃ for 1h and then at 4℃ overnight. Alex Fluor 594 goat antirabbit IgG (Invitrogen, A11037) and Alex Fluor 488 goat anti-mouse IgG (Invitrogen, A11029) were as the secondary antibodyy incubated for 1h at 37℃. Images were selected using a uorescence microscope.
RNA extraction, reverse transcription, and quantitative RT-PCR Total cellular RNA was extracted from B3 cells using the Promega Total RNA Isolation System according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the Access RT-PCR System (Promega). The ROCK1 gene primer pairs for quantitative PCR assay were as follows: sense, 5′-TGTGACTGGTGGTCGGTT-3′ and antisense, 5-′GGTTTTTTGCTTCTTTT′G-3. Primer pairs for β-actin were as follows: sense, 5′-TCGTGCGTGACATTAAGGAG-3′, and antisense, 5′-ATGCCAGGGTACATGGTGGT′-3.
RNA extraction, reverse transcription, and quantitative RT-PCR Total cellular RNA was extracted from B3 cells using the Promega Total RNA Isolation System according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the Access RT-PCR System (Promega). The ROCK1 gene primer pairs for quantitative PCR assay were as follows: sense, 5′-TGTGACTGGTGGTCGGTT-3′ and antisense, 5-′GGTTTTTTGCTTCTTTT′G-3. Primer pairs for β-actin were as follows: sense, 5′-TCGTGCGTGACATTAAGGAG-3′, and antisense, 5′-ATGCCAGGGTACATGGTGGT′-3.
7
Immunofluorescence Imaging of Stem Cell Markers
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Monoclonal antibody against human Oct4A and the secondary anti-mouse IgG HRP antibody was obtained from R&D Systems (Minneapolis, Minnesota, USA). Monoclonal and polyclonal antibodies against human Sox2 and Lin28 were obtained from Cell Signalling Technology (Danvers, Massachusetts, USA). The polyclonal antibody against GAPDH was obtained from IMGENIX (San Diego, CA, USA). Secondary anti-rabbit IgG antibody was obtained from Millipore (Billerica, MA, USA). DAPI Nucleic acid stain and Alex Fluor® 488 goat anti-mouse IgG were obtained from Life Technologies (Carlsbad, CA, USA).
8
Immunofluorescence Analysis of Cardiac Markers
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Cells were fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X-100/PBS and incubated with primary antibodies overnight at 4°C at following dilutions: mouse anti-cTnT (1:200, Lab Vision), mouse anti-MF-20 (1:20 Developmental Studies Hybridoma Bank), rabbit anti-MLC2v (1:200, ProteinTech Group), mouse anti-alpha-Actinin (1:200, Sigma-Aldrich). Cells were then washed and incubated with Alex Fluor 488 goat anti-mouse IgG (1:500, Life Technologies) and/or Alex Fluor 568 goat anti-rabbit IgG (1:500, Life Technologies) for 1 h at RT. Cell nuclei were counter stained with DAPI (1 μg/mL, Sigma-Aldrich). Images were captured using a Zeiss LSM780 confocal microscope, and were analyzed in ZEN 2011 software.
9
Flow Cytometry Analysis of Cardiomyocytes
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Flow cytometry analyses were performed as described previously (Zhang et al., 2012 (link)), with antibody concentrations as following: mouse anti-cTnT (1:200, Lab Vision), mouse anti-MF-20 (1:20 Developmental Studies Hybridoma Bank), and Alex Fluor 488 goat anti-mouse IgG (1:500, Life Technologies). Data were collected and analyzed on a LSRII flow cytometer (Becton-Dickinson).
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