Tissues and cells
Three term placentas (week 38, 40, 41) were collected with the informed, written consent of the mothers after normal vaginal births of healthy children, and dissected into three areas: i.) basal plate area, ii.) intermediate area, and iii.) chorionic plate area. Specimens of app. 1cm3 (and predominantly made up of villous tissue) were rinsed in phosphate buffered saline (PBS) and freshly frozen for Western blot analysis and mRNA isolation for PCR, or fixed for histology and immunohistology as described below. Foreskin specimens from healthy boys were used as positive controls for PCR studies and immunohistology of lymphatic vessels as described . The studies were performed with the informed and written consent of the donors or their legal representatives. Human lymphatic endothelial cells (LECs) were purchased from PromoCell (Heidelberg, Germany), cultured in LEC medium and checked for purity as described recently . All studies on human tissues were approved by the ethics committee of the University Medical Hospital Göttingen, UMG (application no. 18/1/18).
For immunofluorescence studies, specimens were fixed for 20–25 min in 4% paraformaldehyde (PFA), rinsed in PBS, transferred into 10 and 30% sucrose in PBS, and embedded in tissue freeze medium (Tissue Tek, Sakura Finetek Zoeterwoude, NL). Sections of 12 – 14 μm were incubated with the following primary antibodies: Rabbit-anti-human CCBE1 (1:500, Sigma-Aldrich, München, Germany; #R38605), mouse-anti-human CD31 (1:50, BD Pharmingen; clone WM59), mouse-anti-human D2–40/podoplanin (1:200, Dako, Hamburg, Germany; #M3619), rabbit-anti-human LYVE-1 (1:500, ReliaTech, Wolfenbüttel, Germany; #102-PA50AG), rabbit-anti-human PROX1 (1:500, ReliaTech; #102-PA32AG), mouse-anti-human vimentin (1:200, Dako; #GA630), mouse-anti-human CD34 (1:200, Dako; clone QBEnd-10), mouse-anti-human VEGFR-3 (1:100, ReliaTech; #101-M36). Secondary antibodies were: goat-anti-mouse Alexa 488/594 (#A11001; #A21135), goat-anti-rabbit Alexa 594 (#A11012), donkey-anti-goat Alexa 488 (#A11055; MobiTech, Göttingen, Germany). Sections were counter-stained with Dapi and mounted under cover slips with Fluoromount-G (Southern Biotechnology, US). Photos were taken with AxioImagerZ1 (Zeiss, Göttingen, Germany).
Transmission electron microscopy (TEM)
Specimens were fixed with original Karnovsky’s fixative over-night , washed in 0.15 M phosphate buffer for 10 min, transferred into osmium tetroxide solution and incubated for 2 h at 4 °C. Then the samples were rinsed with 0.15 M phosphate buffer for 10 min and subsequently dehydrated in an ascending series of ethanol. Samples were transferred into Epon embedding solution, cut with an Ultracut E microtome (Reichert-Jung) into 90 nm sections. Staining of sections was performed as described . Specimens were analyzed with Leo 906E TEM (Zeiss, Germany).
Cells and tissues were washed 3-times with DPBS (Pan Biotech; Aidenbach, Germany) and 1 mM sodium orthovanadate (Sigma-Aldrich; Steinheim, Germany), homogenized (tissues), and subsequently lysed with RIPA buffer for 10 min on ice. Samples were transferred to micro-tubes (Sarstedt; Nürnberg, Germany) and centrifuged by 20,800x g for 15 min. Protein lysates were collected and used for SDS-PAGE according to standard procedures. Proteins were transferred to PVDF membranes (Roth; Karlsruhe, Germany), which were then washed with TBST (0.1% Tween 20) for 10 min and blocked for 1 h in blocking buffer (5% BSA in TBST 0.1%). Primary anti-human antibodies were: Lyve1 (ReliaTech, Lot 1408R07), PROX1 (Reliatech Lot 0810R19–1), VEGFR-3/FLT4 (ReliaTech, Lot #9D9) and peroxidase-conjugated α-tubulin, 1:10000 (Abcam, ab40742). Incubation was at 4 °C overnight. After washing with TBST (0.1% Tween 20) for 10 min., the peroxidase-conjugated secondary anti-rabbit IgG, 1:2000 (Santa Cruz; USA) were incubated for 1 h. After washing, antigen-antibody complexes were visualized with ECL in Chemidoc Touch Imaging System (Bio-Rad, München, Germany).
Real Time RT-PCR was performed as described . Primers were:
PROX1_fwd: 5′-tgaatccccaaggttctgag-3′, Prox1_rev: 5′-agcagcttgcggagtacatt-3′, LYVE-1_fwd: 5′-gctttccatccaggtgtcat-3′, Lyve-1_rev: 5′-agcctacaggcctccttagc-3′, Podoplanin_fwd: 5′-gaagacatccccagtcctca-3′, Podoplanin_rev: 5′-ctggatggtgctgagacaga-3′. Relative expression was calculated in comparison to LEC expression levels by using the δδCT-Method . Statistical analyses were conducted by two-way-ANOVA with STATISTICA software (Statsoft, Tulsa, Oklahoma). Normal distribution was verified before testing. The standard error of the mean (SEM) was calculated and is shown in the graphs.
Nested PCR was performed to increase specificity and reduce non-specific binding of primers, starting with 40 cycles with outer primers (out) followed by inner primers (in). Primers were:
Prox1_out_fwd: 5′-gtcatctcaccacctgagcc-3′; Prox1_out_rev: 5′-tggaacctcaaagtcatttgct-3′.
Prox1_in_fwd: 5′-gagtgcggcgatcttcaa-3′; Prox1_in_rev: 5′-ggtgacaatccttcctgcat-3′.