Immunostaining sectioned embryos (Conlon lab)

Protocol submitted by VGP from Conlon lab protocols [1]

link to protocol document [2]

Immunostaining of Sectioned Embryos

Wash Buffer: PBS with 1% Triton X-100, 1% Serum

Place slides in a humidity chamber

Wash slides 1x with wash buffer, and apply primary antibody diluted to the appropriate concentration in wash buffer. Only about 300-500 ul of primary antibody per slide is needed. Incubate slides in primary antibody overnight at 4C

Wash slides 3x with wash buffer.

Apply secondary raised against the species of primary antibody at the appropriate concentration diluted in wash buffer. Incubate 30 minutes at room temperature. Protect from light.

Wash 3x.

Apply coverslips, or, staining with multiple antibodies, repeat above procedure with the second primary antibody.

Other stains:

DAPI (from Molecular Probes)-stains DNA in cells- Apply after all antibodies at a concentration of 200ng/mL dissolved in 70% ethanol. 250ul/slide is enough. Incubate 30 minutes at room temperature and protect from light.

Phalloidin (from Molecular Probes. Conjugated to Alexa 488)- stains actin filaments. This can be applied at any point in the process, and only needs to be on for as little as 20 minutes (but overnight is fine too). dilute 1:1000 in wash buffer.

Commonly used antibodies and their dilutions

Mouse anti tropomyosin (CH1- from Developmental Studies Hybridoma Bank)- 1:50

Mouse anti troponin T (CT3- from Developmental Studies Hybridoma Bank)- 1:20

Rabbit anti phospho histone H3 (Upstate)- 1:200

Rabbit anti cleaved caspase 3 (Cell Signaling)- 1:50