Fixation of Embryonic Mouse Tissue for Cytoneme Analysis

Developmental tissue patterning and postdevelopmental homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- time-dependent manner to specify distinct transcriptional programs that instruct reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through the proteins specialized filopodia cytonemes. Cytonemes very thin (≤200 nm diameter) can grow lengths several hundred microns, makes their preservation for fixed-image analysis challenging. This paper describes refined method delicate handling mouse embryos fixation, immunostaining, thick sectioning allow visualization cytonemes using standard confocal microscopy. protocol has been successfully used visualize connect compartments during neural tube development. The technique also be adapted detect across types facilitate interrogation developmental at unprecedented resolution.