Iterative fluorescence imaging

A method for multiplex staining of a biological sample, comprising the steps of:

• a. in a blocking step, contacting a biological sample prepared for imaging with a blocking solution, thereby producing a blocked biological sample, the blocking solution comprising
• i. a blocking compound that is capable of binding to hydrophobic binding sites non-specifically and
• ii. a sulfhydryl-reactive compound;
• b. in a first washing step, contacting said blocked biological sample with a washing solution;
• thereby producing a first washed biological sample;
• c. in a first staining step, contacting said first washed biological sample with a first antibody specific for a first antigen, thereby producing a first stained sample, wherein
• i. said first antibody bears a fluorescent label, or
• ii. said first antibody does not bear a fluorescent label, and subsequent to the first staining step, the first stained sample is contacted with the washing solution and a second staining step is performed, thereby producing a second stained sample, wherein said first stained sample is contacted with a second antibody that
• a. bears a fluorescent label, and
• b. is specific for the first antibody;
• d. in a second washing step, contacting said first stained sample or said second stained sample with the washing solution, thereby producing a second washed sample;
• e. in an imaging step, contacting said second washed sample with an imaging solution at neutral pH containing a radical scavenger, thereby producing an imaged sample and subsequently recording a fluorescence image of the second washed sample, thereby producing an imaged sample wherein the radical scavenger of the imaging solution is also an acceptor for free radical-induced photo-crosslinking;
• f. in a third washing step, contacting said imaged sample with the washing solution, thereby producing a third washed sample;
• g. in an elution step, contacting said third washed sample with an elution solution thereby producing an eluted sample, wherein the elution solution
• i. is characterized by a pH of about 2.2 to 4.0 and
• ii. comprises a buffering component, a reducing agent and at least one compound disrupting hydrogen bonds;
• wherein steps a to g are repeated 1 or more times, employing a different first antibody in each repeat.