1. Methods in Developmental Biology Research : In Situ Hybridization and Immunohistochemistry

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

3. 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

5. 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

7. 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

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

9. 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

10. FISH: Detecting Genomic Sequence on Chromosomes

11. FISH: X and Y Chromosome Markers in the Prostate

13. 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

14. 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.

15. Immunohistochemistry : Metastatic Rhabdoid Carcinoma
Look for a brown precipitate!