Envision hrp labelled polymer k4003

After euthanasia, the hindlimbs of the mice were cut off and fixed in 4% buffered formaldehyde for 48 h. The feet were then cut 1 cm distal to the heel and decalcified in 4.0 M formic acid/0.5M sodium formate for 24 h. The limbs were then embedded in paraffin. We stained 3 μm thick sections with anti-LYVE1 (ab33682; Abcam, Cambridge, UK). Tris-EGTA buffer (pH 9.0) at 60 °C overnight was used to achieve antigen retrieval. Specimens were incubated with primary antibody (1:10,000) for 60 min at room temperature. Envision + HRP labelled polymer (K4003; Dako; Agilent, Glostrup, Denmark)/DAB + was used as detection system.

Hind limbs were fixed in 4% paraformaldehyde for 48 hours. Afterwards the feet were cut 10mm distal to the heel and decalcified in 4.0M formic acid/0.5M sodium formate for 4hours before paraffin embedding. To detect lymphatic vessels, 3µm thick sections were obtained from the cut surface and stained using anti-LYVE1 (ab33682; Abcam, Cambridge, UK) as primary antibody was used. Antigen retrieval was achieved by Tris-EDTA buffer (pH 9.0) microwaving for 15min at 440W. Specimens were incubated with primary antibody (1:1000) for 60min at room temperature.
Envision+ HRP labelled polymer (K4003; Dako; Agilent, Glostrup, Denmark) was used as detection system.