Dissection and Immunostaining of Lymph Glands

Lymph glands were dissected and processed as previously described [11 (link)]. The following primary antibodies were used: mouse anti-Col [10 (link)]; anti-proPO (1/200; T. Trenczek, Justus-Liebig-University Giessen, Giessen, Germany); anti-P1 (1/30; I. Ando, Institute of Genetics, Biological Research Center of the Hungarian Academy of Science, Szeged, Hungary); anti-Antp (1/100) and anti-Hindsight (Hnt)/pebbled (peb) (1/100) (Developmental Studies Hybridoma Bank). Rabbit anti-H3P (1/200; Upstate Biotechnology); anti-αPS4 (1/200, [11 (link)]. For in situ hybridization, DIG-labeled antisense RNA probes against col [10 (link)], tep4 [11 (link)] and latran (lat)/eye transformer (ey) [34 (link)] were used. In situ hybridized samples were mounted in 1XPBS-60% glycerol. Immunostained samples were mounted in Vectashield medium (Vector Laboratories) and analyzed with laser scanning confocal microscopy (Leica SP5). Cell nuclei were visualized using the dye TOPRO3 (Molecular Probes).