Download the protocol (PDF)
Go to the Immunofluorescence Tested Antibodies web page
Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
Triton X-100 Stock Solution - 10% (v/v) solution in PBS
Antibody Diluent - 10% DCS in PBS
Hoechst 33342 Stock Solution - 10 mg/mL in PBS
Permeabilization and Blocking Solution - 0.2% Triton X-100 and 10% Donor Calf Serum in PBS
Primary Antibody Solutions – Prepare 8 different primary antibody dilutions ranging from 1:50 to 1:6,400 using Antibody Diluent.
Secondary Antibody Solution – dilute secondary antibody 1:1,000 using Antibody Diluent. Add Hoechst 33342 Stock Solution to a final concentration of 1 mg/mL (10,000-fold dilution of Stock Solution).
All images were acquired using the PerkinElmer® Opera® high-throughput confocal imager. All antibodies were imaged at 8 different dilutions ranging from 1:50 to 1:6400 with 2–3 images taken per well. Plates were imaged at both 20´ (for data acquisition) and 40´ (for high quality image generation). Antibody signal was measured using the 640 nm (red) laser and Hoechst intensity was measured using the 405 nm (violet) laser. In both cases, the exposures were taken separately for each channel to limit fluorescent bleedthrough between the channels. Exposure times varied for each channel with maximums of 2,000 ms for the 640 nm channel and 400 ms for the 405 nm channel.
Data was generated using the PerkinElmer Acapella® software package. Intensity values were obtained using the NFkB Translocation algorithm. Nuclear intensity was calculated from the antibody signal in the area overlaid with Hoechst staining. Cytoplasmic intensity was calculated from a cytoplasmic ring region defined by the area outside of the nuclear region. All values represent the average intensity per cell in each field.
Triton is a registered trademark of Dow Chemical Company.
PerkinElmer, Opera, and Acapella are registered trademarks of PerkinElmer Inc.
We warrant that our products conform to the information contained in this and our other publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of the invoice or packing slip.