SCp2 mammary epithelial cells with MMP3 knock down and control were cultured for 72 h on glass coverslips, fixed with 4% paraformaldehyde (15710, EMS) in 1× PBS for 10 min, washed with 1× PBS, and permeabilized in 0.25% Triton X-100 (t8532, Sigma-Aldrich) in 1× PBS for 10 min. Cells were then blocked with 2% bovine serum albumin (A7888, Sigma-Aldrich) for 1 h, followed by incubation with a goat polyclonal antibody targeting MMP3 (ab18898, Abcam) in blocking buffer overnight at 4 °C and donkey α-goat secondary antibody (A11058, Life Technologies) for 1 h at room temperature. DNA DAPI (D9542, Sigma-Aldrich) was used to stain cell nuclei. Fluorescence images were acquired with an upright confocal microscope (LSM710, Zeiss) using a 1.4 NA 63× oil immersion.
Dna dapi
Manufactured by Merck Group
DNA DAPI is a fluorescent dye used in laboratory applications. It selectively binds to the adenine-thymine (A-T) rich regions of DNA, allowing for the visualization and detection of DNA in various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
A11058, by Thermo Fisher Scientific (1 mentions)
T8532, by Merck Group (1 mentions)
A7888, by Merck Group (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rabbit anti γ tubulin, by Merck Group (1 mentions)
Rat anti e cadherin, by Thermo Fisher Scientific (1 mentions)
Mouse anti p63, by Santa Cruz Biotechnology (1 mentions)
Chick anti gfp, by Abcam (1 mentions)
Mouse anti acetylated tubulin, by Merck Group (1 mentions)
Fluoromount, by Merck Group (1 mentions)
2 protocols using dna dapi
1
Immunostaining of MMP3 in Mammary Epithelial Cells
2
Immunofluorescence Analysis of Airway Cells
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Cultured embryonic trachea were fixed 1 h at 4°C in 4% paraformaldehyde in PBS (4% PFA), sucrose protected and embedded in Optimal Cutting Temperature medium (OCT; Tissue Tek, Leiden, The Netherlands) before cryosectioning at 10 μm. Cells in 24-well transwell inserts were fixed in 4% PFA for 20 min at room temperature and stained whole mount with a 15 min 0.3% TritonX-100 permeabilisation step. Membranes were mounted in the centre of a SecureSeal™ image spacer (Sigma-Aldrich, GBL654006). Mounting medium was Fluoromount (Sigma-Aldrich).
Primary antibodies: mouse anti-acetylated tubulin (1:3000, Sigma-Aldrich, clone 6-11B-1); rat anti-E-cadherin (1:3000, Invitrogen, clone ECCD-2); mouse anti-Foxj1 (1:300, BD Bioscience, clone 2A5); rabbit anti-γ-tubulin (1:1000, Sigma-Aldrich, clone 310381); chick anti-GFP (1:1000, Abcam ab13970); mouse anti-p63 (1:500, Santa Cruz Biotechnology, clone 4A4); rabbit anti-Scgb1a1 (1:500; Santa Cruz Biotechnology, sc-25555). Antigen retrieval was in 10 mM boiling sodium citrate buffer pH6 for Foxj1 and p63 on cryosections. Alexa Fluor-conjugated secondary antibodies (ThermoFisher Scientific) were used at 1:2000. DNA (Dapi, Sigma-Aldrich).
Primary antibodies: mouse anti-acetylated tubulin (1:3000, Sigma-Aldrich, clone 6-11B-1); rat anti-E-cadherin (1:3000, Invitrogen, clone ECCD-2); mouse anti-Foxj1 (1:300, BD Bioscience, clone 2A5); rabbit anti-γ-tubulin (1:1000, Sigma-Aldrich, clone 310381); chick anti-GFP (1:1000, Abcam ab13970); mouse anti-p63 (1:500, Santa Cruz Biotechnology, clone 4A4); rabbit anti-Scgb1a1 (1:500; Santa Cruz Biotechnology, sc-25555). Antigen retrieval was in 10 mM boiling sodium citrate buffer pH6 for Foxj1 and p63 on cryosections. Alexa Fluor-conjugated secondary antibodies (ThermoFisher Scientific) were used at 1:2000. DNA (Dapi, Sigma-Aldrich).
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