A method of determining the abundance and spatial position of at least two target analytes in a biological sample, wherein the biological sample is prepared by: i) contacting the biological sample with at least one nucleic acid probe by incubating a mounted biological sample with a solution comprising a plurality of in situ hybridization (ISH) probes, wherein the solution comprises at least two species of ISH probes, wherein at least one species of ISH probe comprises a unique target binding domain that binds to one of the at least two target analytes and a unique barcode domain specific for the one target analyte, wherein the barcode domain comprises at least one attachment region; and ii) washing the mounted biological sample, the method comprising: a) contacting the prepared biological sample with a plurality of reporter probes, wherein each reporter probe comprises at least one detectable label, thereby hybridizing a reporter probe to an attachment region of the barcode domain of at least one ISH probe hybridized to a target analyte in the prepared biological sample; b) removing non-hybridized reporter probes from the prepared biological sample; c) recording the identity and spatial position of the at least one detectable label of the hybridized reporter probes; d) removing the at least one detectable label of the hybridized reporter probes; and e) repeating steps (a)-(d) until each attachment region in the barcode domains of the ISH probes hybridized to a target analyte in the prepared biological sample have been hybridized to a reporter probe comprising at least one detectable label, thereby determining the abundance and spatial position of the at least two target analytes in the biological sample based on the sequence in which the detectable labels were recorded. i) contacting the biological sample with at least one nucleic acid probe by incubating a mounted biological sample with a solution comprising a plurality of in situ hybridization (ISH) probes, wherein the solution comprises at least two species of ISH probes, wherein at least one species of ISH probe comprises a unique target binding domain that binds to one of the at least two target analytes and a unique barcode domain specific for the one target analyte, wherein the barcode domain comprises at least one attachment region; and wherein the solution comprises at least two species of ISH probes, wherein at least one species of ISH probe comprises a unique target binding domain that binds to one of the at least two target analytes and a unique barcode domain specific for the one target analyte, wherein the barcode domain comprises at least one attachment region; and ii) washing the mounted biological sample, a) contacting the prepared biological sample with a plurality of reporter probes, wherein each reporter probe comprises at least one detectable label, thereby hybridizing a reporter probe to an attachment region of the barcode domain of at least one ISH probe hybridized to a target analyte in the prepared biological sample; b) removing non-hybridized reporter probes from the prepared biological sample; c) recording the identity and spatial position of the at least one detectable label of the hybridized reporter probes; d) removing the at least one detectable label of the hybridized reporter probes; and e) repeating steps (a)-(d) until each attachment region in the barcode domains of the ISH probes hybridized to a target analyte in the prepared biological sample have been hybridized to a reporter probe comprising at least one detectable label, <EOS>