Paraffin embedded slides were deparaffinized using five-minute washes in xylenes (x2), 100% ethanol, 95%, 80% 70% ethanol, and PBS. Antigen retrieval was conducted for 20 minutes in boiling citrate buffer (pH 6, Sigma). Slides were cooled and then blocked in 5% goat serum (Sigma Aldrich) in PBS with 0.05% Tween-20 for 30 minutes. Primary antibody against Ki-67 (Abcam, ab16667, 1:1000) was diluted in blocking solution and samples were incubated overnight at 4°C. Slides were washed three times with PBS-Tween prior to incubation with fluorescence-conjugated secondary antibody (Invitrogen, 1:200) in blocking solution for 1 hour at room temperature. Following 3 PBS-Tween washes with the final wash containing 5 ug/mL DAPI nuclear stain (Roche), samples were dried and mounted using ProlonGold Antifade (Invitrogen). Fluorescence intensity was measured on a Zeiss inverted LSM 780 laser scanning confocal microscope at the Bioimaging Resource Center at Rockefeller University. Images were analyzed using ImageJ, by calculating mean fluorescence intensity of sample to normalized DAPI signal.
Prolongold antifade
Manufactured by Thermo Fisher Scientific
ProlonGold Antifade is a mounting medium designed to preserve fluorescent signals in microscopy samples. It helps maintain the brightness and stability of fluorophores, allowing for longer observation and imaging.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Goat serum, by Merck Group (2 mentions)
Citrate buffer, by Merck Group (2 mentions)
Inverted lsm 780 laser scanning confocal microscope, by Zeiss (2 mentions)
Ab16667, by Abcam (2 mentions)
Dapi nuclear stain, by Roche (2 mentions)
2 protocols using prolongold antifade
1
Immunofluorescence Staining of Ki-67
2
Immunofluorescence Staining of Ki-67
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Paraffin embedded slides were deparaffinized using five-minute washes in xylenes (x2), 100% ethanol, 95%, 80% 70% ethanol, and PBS. Antigen retrieval was conducted for 20 minutes in boiling citrate buffer (pH 6, Sigma). Slides were cooled and then blocked in 5% goat serum (Sigma Aldrich) in PBS with 0.05% Tween-20 for 30 minutes. Primary antibody against Ki-67 (Abcam, ab16667, 1:1000) was diluted in blocking solution and samples were incubated overnight at 4°C. Slides were washed three times with PBS-Tween prior to incubation with fluorescence-conjugated secondary antibody (Invitrogen, 1:200) in blocking solution for 1 hour at room temperature. Following 3 PBS-Tween washes with the final wash containing 5 ug/mL DAPI nuclear stain (Roche), samples were dried and mounted using ProlonGold Antifade (Invitrogen). Fluorescence intensity was measured on a Zeiss inverted LSM 780 laser scanning confocal microscope at the Bioimaging Resource Center at Rockefeller University. Images were analyzed using ImageJ, by calculating mean fluorescence intensity of sample to normalized DAPI signal.
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