Antibody Staining of Scale Melanocytes: A Technique to Detect Transcription Factors Expressed in Dorsal Scale-associated Melanocytes of Zebrafish Models
Transcripción
– Transcription factors are proteins containing DNA-binding domains. They bind to specific DNA sequences and help regulate gene expression in cells. To detect transcription factor expression in scale-associated melanocytes, first, harvest dark pigmented dorsal scales from an anesthetized zebrafish. Fix the scales and treat them with a permeabilization solution to render the individual melanocytes permeable.
Treat with a bleach solution to remove the dark melanin pigment granules from the cells. This bleaching process eliminates the chances for pigment-associated background signals during the subsequent staining steps. Incubate with a blocking solution containing proteins to block non-specific protein binding sites on the cell surface.
Now, treat the sample with the desired primary antibodies. These antibodies bind to their target transcription factors within the cells. Next, label the sample with fluorescent-tagged secondary antibodies specific to the primary antibodies. Additionally, treat the sample with an appropriate fluorescent DNA binding dye to stain the cell nuclei. Use a fluorescence microscope to detect bright fluorescent emissions from the transcription factor-antibody complexes and cell nuclei.
In the following protocol, we will show the immunostaining of dorsal scale-associated melanocytes expressing melanocyte-inducing transcription factor-A, or mitfa, in a transgenic zebrafish.
– To perform immunostaining, anesthetize the fish in 0.17 milligrams per milliliter of Tricaine. With incident light under a dissecting microscope, use sharp fine-tipped forceps to pluck dorsal scales, taking care not to damage the melanocyte-containing half of the scale. Place the scales directly from the forceps into 1 milliliter of fixative and incubate at room temperature, while turning, for greater than or equal to 2 hours. Transfer the fish into fish water and monitor the fish to confirm successful recovery.
To bleach pigmented melanocytes, first, wash fixed scales in PBST two times for 5 minutes at room temperature. Next, add 1 milliliter of freshly made bleach solution and cover with parafilm or cap locks to prevent gas from bursting tubes open. Continue bleaching until pigment is gone. Then, wash in PBST four times for 5 minutes at room temperature.
Incubate for at least 30 minutes in block solution. Then, incubate with about 400 microliters of primary antibody in block solution overnight at room temperature. The following day, wash the scales three times for 30 minutes in PBST at room temperature. Incubate with secondary antibody for 2 hours to overnight at room temperature.
After staining with DAPI, apply a drop of vectashield to a glass slide and mount the scales so that the concave side of the scales faces down against the slide. Place a glass cover slip over the samples. Use clear nail polish to seal the edges and observe the slides under a fluorescence microscope.
