Whole mount immunohistochemistry (WM-IHC)
Marta Wawrzyniak, Nathalie Weber, Simon Blanchoud
Abstract
This protocol has been successfully used with colonial tunicates has been adapted to these animals based on the following publication:
Steps
Fixation
Clean the slide with the colony of your interest.
Anesthetize the animals following this protocol. See Whole colony fixation V.1.
Fix the animals in 4% PFA at Room temperature . See Whole colony fixation V.1.
Wash twice in 3.3x PBS for 0h 30m 0s .
Rinse quickly 3 times with 1x PBS and then leave in 1x PBS for 1h 0m 0s .
Permeabilization and quenching
Place samples in PBST-SC.
Shake at Room temperature .
Rinse once with 1x PBS.
Blocking and staining
Place samples in PBST-GS.
Shake at Room temperature for 2h 0m 0s .
Stain your samples with primary antibody for 30h 0m 0s .
Dilute the primary antibody in PBST-GS according to the manufacturer.
On the slide, circle the system of interest with a PAP pen and wait 0h 2m 0s for the circle to dry.
Place the slide flat into the humidity chamber.
Pipette the primary antibody solution on the circled system and incubate at 4°C for 30h 0m 0s .
Wash four times in 1x PBST at Room temperature for 0h 30m 0s slowly on a linear.
Stain your samples with secondary antiboby for 30h 0m 0s .
Dilute the antibody in PBST-GS according to the manufacturer (keep solution and sample protected from the light!).
Incubate in darkness at 4°C in the humidity chamber.
Wash four times in 1x PBST at Room temperature for 0h 30m 0s slowly on a linear shaker.
Dry as much as possible.
Circle the sample with vaseline to build a custom-made chamber.
Mount with mounting medium, cover with a coverslip.
Image your slides directly or store them at 4°C .
