For immunocytochemistry of active integrin‐β1, cells were seeded on sterile coverslips in a six‐well plate at a density of 173,000 cells per well. The coverslips were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X‐100 on ice for 1 min. After blocking in 5% BSA‐1X PBS, active integrin‐β1 antibody (1:100) was added for 1 h followed by 1h incubation with secondary antibody. After repeated washing in 1X PBS, cells were stained with the DNA stain DAPI (4,6‐diamidino‐2‐phenylindole dihydrochloride; #1023627001; Roche) and mounted using Prolong gold anti‐fade mount media (#P36930; Invitrogen) on glass slides. Imaging and z‐stacks (1‐µm z‐slice) were acquired using a Leica TCS SPEII confocal microscope maintaining consistent acquisition parameters between experiments.
Prolong gold anti fade mount media
Manufactured by Thermo Fisher Scientific
Prolong Gold anti-fade mount media is a commercially available product designed to be used as a mounting medium for fluorescently labeled samples in microscopy applications. It is formulated to help preserve and protect fluorescent signals from photobleaching.
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5 protocols using prolong gold anti fade mount media
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Immunocytochemistry of Active Integrin-β1
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Immunocytochemistry of Active Integrin-β1
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Immunocytochemistry of Fibronectin
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For immunocytochemistry of FN, cells were seeded on sterile coverslips in a 6-well plate at a density of 173,000 cells per well. The coverslips were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 on ice for 1 min. After blocking in 5% BSA-1 × PBS, FN antibody (1:70) was added for 1 h followed by 1 h incubation with secondary antibody. After repeated washing in 1 × PBS, cells were stained with the DNA stain DAPI (4,6-diamidino-2-phenylindole dihydrochloride) (Roche #1023627001) and mounted using Prolong gold anti-fade mount media (Invitrogen #P36930) on glass slides. Imaging and z-stacks (1 µm z-slice) were acquired using a Leica TCS SPEII confocal microscope at consistent acquisition parameters for each experiment.
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Immunostaining of Mammary Acini
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Immunostaining of Acini for Integrin-α6 and FN
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Immunostaining of acini was performed as previously described.26 Briefly, media was aspirated from each well of the chamber slide and acini were fixed with 4% paraformaldehyde for 20 min followed by permeabilization for 10 min at 4°C in 0.5% Triton‐PBS. After repeated rinsing in PBS‐glycine, blocking solution (130‐mM NaCl, 7‐mM Na2HPO4, 3.5‐mM NaH2PO4, 7.7‐mM NaN3, 0.1% BSA, 0.2% Triton X‐100, 0.05% Tween‐20, and 10% goat serum) was added for 45–60 min at room temperature. The acini were subsequently incubated with integrin‐α6 primary antibody or FN primary antibody (1:100) overnight at 4°C followed by secondary antibody incubation (Alexa 568) in blocking buffer. The nuclei were stained with DAPI (#1023627001; Roche) and mounted using Prolong gold anti‐fade mount media (#P36930; Invitrogen) on glass slides. Imaging and z‐stacks (4‐µm z‐slice) were acquired using a Leica TCS SPEII confocal microscope maintaining consistent acquisition parameters between experiments.
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