A method for in situ imaging of samples by cycle multiplexing comprising the steps of: (i) providing a sample immobilized on a sample support; (ii) providing a microfluidic device comprising a microfluidic chamber, at least one fluid inlet at one end of said microfluidic chamber and at least one fluid outlet at another end of said microfluidic chamber configured to conduct a fluid supplied from a fluid feeding system under pressure through the microfluidic chamber for advective transport of fluidic substances and reagents inside said microfluidic chamber in a uniform manner, wherein at least one wall of the microfluidic chamber is formed by the sample support and wherein the volume of the microfluidic chamber is from 2.5 and 200 μl; (iii) mounting said sample support over said microfluidic chamber with the sample facing the inside of the microfluidic chamber; (iv) injecting in sequence a plurality of reagents, including at least one imaging probe, through the fluid inlet into the microfluidic chamber, at a flow rate in a range between 1 μl/s and 100 μl/s; (v) imaging a signal emitted by components of the sample reacted with said at least one imaging probe; (vi) repeating steps (iv) and (v) with different imaging probes; wherein said injecting in sequence a plurality of reagents includes: - an elution step where an elution buffer is injected for removing undesirable material potentially remaining on the sample; (S1) - a non-specific binding blocking step where a blocking buffer is injected; (S2) - a sample labelling step where an imaging probe is injected; (S3) and - an optional pre-imaging step where an imaging buffer is injected, wherein each of these steps may be preceded and/or followed by an optional washing step wherein a washing buffer is injected, (S4) wherein said at least one imaging probe results from the injection of a sequence of specific antibodies as labelling probes and chromogen or fluorescent detection molecules, targeting the molecular entities to be analyzed within the said sample. (i) providing a sample immobilized on a sample support; (ii) providing a microfluidic device comprising a microfluidic chamber, at least one fluid inlet at one end of said microfluidic chamber and at least one fluid outlet at another end of said microfluidic chamber configured to conduct a fluid supplied from a fluid feeding system under pressure through the microfluidic chamber for advective transport of fluidic substances and reagents inside said microfluidic chamber in a uniform manner, wherein at least one wall of the microfluidic chamber is formed by the sample support and wherein the volume of the microfluidic chamber is from 2.5 and 200 μl; (iii) mounting said sample support over said microfluidic chamber with the sample facing the inside of the microfluidic chamber; (iv) injecting in sequence a plurality of reagents, including at least one imaging probe, through the fluid inlet into the microfluidic chamber, at a flow rate in a range between 1 μl/s and 100 μl/s; (v) imaging a signal emitted by components of the sample reacted with said at least one imaging probe; (vi) repeating steps (iv) and (v) with different imaging probes; - an elution step where an elution buffer is injected for removing undesirable material potentially remaining on the sample; (S1) - a non-specific binding blocking step where a blocking buffer is injected; (S2) - a sample labelling step where an imaging probe is injected; (S3) and - an optional pre-imaging step where an imaging buffer is injected, wherein each of these steps may be preceded and/or followed by an optional washing step wherein a washing buffer is injected, (S4) <EOS>