Methods in Developmental Biology Research : In Situ Hybridization and Immunohistochemistry

Immunohistochemistry and in situ hybridization allow researchers to pinpoint the expression of their protein and nucleic acid targets, respectively.

Immunohistochemistry and in situ hybridization: Allow for specific detection of targets within tissues while maintaining the morphology of the tissue Identify specific cell types that are expressing the target, as well as, when the cell type is expressing that target Can be done in retrospect with paraffin-embedded specimens

In Situ Hybridization Allows for the detection of nucleic acid sequences in cells and tissues shows where, when, and in which tissues a specific gene is being expressed can use both RNA and DNA probes can utilize both tissue sections or whole embryos

Choosing probes for in situ Antisense probe complementary strand to the mRNA should hybridize to the target mRNA Sense probe same sequence as the mRNA should not hybridize to the target mRNA can serve as a negative control

How do you “see” your probe? ATCCGATGCATCCGA Fluorescence ATCCGATGCATCCGA Radioactivity ATCCGATGCATCCGA Digoxigenin + Labeled Antibody DIG DIG DIG DIG

In Situ: Whole Mount vs. Sectioned Embryos Whole mount in situ for Sonic Hedgehog in mouse embryos Heart section in situ for TBX5 in mouse Whole Mount in Situ

FISH: Detecting Genomic Sequence on Chromosomes

FISH: X and Y Chromosome Markers in the Prostate

Immunohistochemistry Allows for the detection of target antigens within tissue (usually proteins) Utilizes antibodies to determine protein expression - detects target within specific cells, gives a relative level of expression, and subcellular localization - utilizes tissue sections and whole embryo mounts Tracheal marker in Drosophilia embryo

Primary and secondary antibodies are used to detect protein of interest Primary antibody recognizes epitope of interest (protein or protein with modification) Secondary antibody recognizes first antibody and includes a marker visible under the microscope Markers may be fluorescent or appear as a brown precipitate depending on method used.

Immunohistochemistry : Metastatic Rhabdoid Carcinoma Look for a brown precipitate!