4i technology

This section explains in a step-by-step protocol how to perform 4i on your sample.

General Protocol
  1. Grow your cells to desired confluency.
  2. Fix cells at 4% Paraformaldehyde (in PBS) for 30min.
  3. Thoroughly wash cells with PBS.
  4. Permeabilize cells with 0.5% Triton X-100 for 15min.
  5. Thoroughly wash cells with ddH2O.
  6. Incubate cells with EB for 30min at room temperature.
  7. Thoroughly wash cells with PBS.
  8. Add sBS to sample and incubate for 1h at room temperature.
  9. Thoroughly wash cells with PBS.
  10. Perform immunofluorescence protocol against antigens of interest. Use cBS to dilute antibodies. Incubate primary antibodies for 2h at room temperature. Thoroughly wash cells with PBS. Incubate secondary antibodies for 1h at room temperature.
  11. Thoroughly wash cells with PBS.
  12. To stain the nuclei of cells, add DAPI at a final concentration of 400ng/ml to your cells and incubate for 15min at room temperature.
  13. Thoroughly wash cells with ddH2O.
  14. ​Add Imaging buffer to cells.
  15. Sample is ready to be imaged. Image.

​Repeat steps 5 to 15 to generate multiplexed 4i images of your cells.
​Enjoy!
​
Protocol adapted to multiwell plates (384- and 96-well plates) can be downloaded below or in the Downloads section.
Downloads
Protocol_4i_Gutetal_384wellplate.pdf
File Size: 32 kb
File Type: pdf
Download File

 Schematic reptesentation of the 4i protocol

Materials needed:
Elution Buffer (EB) used to elute antibodies from sample
500 mM L-glycine, 3 M urea, 3 M guanidinum hydrochloride, 70 mM TCEP-HCl in ddH2O.
Add TCEP-HCl only before adjust before using EB. Then adjust to pH 2.5 and use.

4i Blocking Solution (sBS) used to block sample
1% bovine serum albumin (BSA), 100 mM ammonium chloride, 150 mM maleimide in phosphate buffered saline (PBS).
Add maleimide and immediately before using sBS.

Conventional Blocking Solution (cBS) used for antibody hybridization
1% BSA, 100 mM ammonium chloride in PBS.

Imaging Buffer (IB) used to prevent photocrosslinking during imaging
700 mM N-Acetyl-Cysteine in ddH20.
Adjust IB to pH 7.4

Paraformaldehyde
Triton X-100
DAPI (4',D-diamidino-2-phenylindole)

1x Microscope