This section explains in a step-by-step protocol how to perform 4i on your sample.
Repeat steps 5 to 15 to generate multiplexed 4i images of your cells.
Protocol adapted to multiwell plates (384- and 96-well plates) can be downloaded below or in the Downloads section.
Schematic reptesentation of the 4i protocol
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