Hi, I am doing the sprouting assay with the mouse thoracic duct rings (TD rings) in the Matrigel. I’ve already tried several times but never succeeded. I’ve been trapped in some troubles:

a). I’m not sure whether the tissue I harvested was from TD.

I opened the thoracic cavity of the mouse and removed lungs, finding the descending aorta first. Then I carefully removed the adipose tissue around the aorta. Bluntly dissect the transparent “thoracic duct” that is posteriomedial to the aorta. At least I thought it should be the duct. The adipose tissue around the TD was also carefully removed with microsurgical forceps. I kept moisture of tissues through the operation and used microsurgical instruments under microscope.

b). No real TD sprouts grew in the Matrigel (up to 15 days).

There were always some suspected branches protruding from the TD rings. But they’ve been proved by IHC not the true lymphatic sprouts. I guess they might be smooth muscle cells or fibroblasts. I can’t say what they really were.

c). When I did the whole-mount IHC of these rings, I got very weak green fluorescence every time. The fluorescence strength was a bit stronger under 40x magnification than 10x, although it was still quite weak. I don’t know why this happened in various magnification folds. I don’t know either whether those weak images could mean positive.

My IHC protocol: wash > fixation with 4% PFA for 30min > wash > permeabilization by 0,1% tritonx-100, 10min, wash > block at RT, 1h > 1:100 anti-LYVE1-ab. (add only dilution buffer for the negative control rings), 4℃, overnight > wash > 1:1000 Alexa 488, 37℃ dark incubation, 1h > wash > DAPI counter stain 10min > wash > fluor. Microscope (The background fluorescence in the negative control rings would be subtracted). Both the primary and secondary antibodies were newly bought and properly aliquoted and stored.

I would appreciate any help that may clear my confusions!