Alexa fluor 594 anti rat

maRTA was performed as previously described with some modifications [31 (link)]. Briefly, 36 hours after siRNA transfection, HeLa cells were pulse-labeled with 50 μM iododeoxyuridine (IdU) for 40 min. Cells were then treated or not with 2 mM HU for 5 hours or 16 hours. The cells were released in fresh medium containing 50 μM of chlorodeoxyuridine (CldU) for 40 min. Cells were then harvested and embedded into agarose plugs containing 20,000 cells/plug. After proteinase K digestion and agarose digestion by beta-agarase, DNA fibers were stretched on 3-aminopropyltriethoxysilane coated slides (LabScientific) using polydimethylsiloxane molds fashioned with micro-capillary channels prepared as described [31 (link)]. DNA fibers were then denatured in 2.5 M HCl, and probed with the following antibodies: mouse IgG₁ anti-BrdU/IdU (clone BD44, Becton Dickinson), rat anti-BrdU/CldU (clone B1/75, Bio-Rad OBT0030), and mouse IgG_2a anti-ssDNA (clone 16–19, Millipore). Secondary antibodies included Alexa Fluor 488 anti-mouse IgG₁, Alexa Fluor 594 anti-rat, and Alexa Fluor 647 anti-mouse IgG_2a, respectively (Life Technologies). Images were acquired on Leica DMI6000 epifluorescence microscope using Leica LAS-AF software. Signals were measured using NIH ImageJ software with custom-made modifications and the data analyzed with GraphPad Prism software.

For the BrdU immuno-labeling, the brain sections were rinsed three times in 0.1 M PBS and incubated in 2 N HCl at 37°C for 15 min, followed by 0.1 M borate buffer (pH = 8.6) for 10 min and washed 3× with 0.1 M PBS. Sections were incubated in blocking solution for 40 min. Then, sections were incubated with some combinations of the following primary antibodies: rat anti-BrdU (1:500; AbD Serotec Cat # OBT0030, RRID:AB_609568), mouse anti-NeuN (1:500; Millipore Cat # MAB377, RRID:AB_2298772), rabbit anti-GFAP (1:100; Dako Cat # Z0334, RRID:AB_10013382), rabbit anti-Sox2 (1:500, Abcam Cat # AB97959; RRID:AB_2341193), guinea pig anti-doublecortin (DCX, 1:1000: Millipore Cat# AB2253, RRID:AB_1586992) in blocking solution at 4°C overnight. Sections were rinsed 3× with 0.1 M PBS, and incubated in 0.1 M PBS containing 10% fetal bovine serum and conjugated secondary antibodies (Alexa Fluor^® 488 anti-rat Cat # A-21208; Alexa Fluor^® 594 anti-rat Cat # A-11007; Alexa Fluor^® 488 anti-mouse Cat # A32723; Alexa Fluor^® 594 anti-rabbit; Alexa Fluor^® 594 Cat# R37117; anti-guinea pig Cat# A-11076; dilution 1:1000; Thermo Fisher) for 1 h at room temperature and washed 3× with 0.1 M PBS. Nuclear counterstaining was done with 4′,6-diamidino-2-phenylindole (DAPI; Abcam Cat # ab104139, Cambridge, MA, USA).

Xenograft tumour tissue was cryo-sectioned (20 µm) and processed as previously described.^{6 (link)} Endothelial cells were stained with a monoclonal Armenian-hamster anti-mouse CD31 antibody (1:200, Abcam) or a fluorescein isothiocyanate (FITC)-labelled anti-CD31 antibody (1:200, clone 390, Biolegend), and apoptotic cells were detected with a rabbit anti-cleaved caspase-3 (Asp175) antibody (1:200, Cell Signaling). Pericytes were detected either by a rabbit anti-NG2 Chondroitin Sulphate Proteoglycan antibody (1:100, Millipore) or a rat anti-PDGF-R antibody (1:100, CD140b, eBioscience). Vascular endothelial (VE) cadherin was stained with a rat anti-CD144 (VE-cadherin) antibody (BD Pharmingen). The following secondary antibodies derived from goat were used to label the respective primary antibodies with fluorescent dyes: Alexa Fluor 647 anti-hamster, Alexa Fluor 594 anti-rat, Alexa Fluor 594 anti-rabbit and Alexa Fluor 488 anti-hamster (1:500, Thermo Fisher Scientific). Vascular leakage was quantified by analysing intravenously injected FITC-dextran (molecular weight 2000 kDa, Sigma), pimonidazole adducts were detected with a FITC-labelled anti-pimonidazole antibody to visualise hypoxia; both procedures have been previously described in detail^{6 (link),19 (link)}; nuclei were stained with 4,6-diamidin-2-phenylindol (DAPI).

Mice were euthanised, and diaphragms and ears were harvested. The tissues were subsequently washed, blocked in immunomix-2 (IM-2) containing 0.1% Triton-X (Honeywell), 1% BSA/PBS (Merck) and 0.5% normal donkey serum (Merck). Both tissues were subsequently incubated overnight at 4 °C in IM-2 with rat anti-MECA-32 antibody (5 µg/ml, Biolegend, San Diego, USA).
The following day, the tissues were incubated for 3 h with Alexa Fluor-conjugated secondary antibodies, i.e. Alexa Fluor 594 anti-rat and Alexa Fluor 488 anti-rabbit (3 µg/ml, Invitrogen, Basel, Switzerland). Diaphragms were subsequently fixed in 4% PFA for 2 h at 4 °C. Samples were washed with PBS and mounted using Mowiol (Vector Laboratories, Burlingame, USA).
Tissues were analysed on a Zeiss LSM780 inverted confocal microscope (Carl Zeiss, Oberkochen, Germany) using a 10 × 0.3 NA EC Plan-Neofluar objective and processed with the Imaris software (version 7.1.1; Bitplane, Zurich, Switzerland). In the diaphragm, images of LYVE-1⁺ and MECA-32⁺ vessels were taken in the middle part of central tendon where the distribution of both lymphatic and blood vessels is homogenous and reproducible. In the ear skin, lymphatic and blood vessels were imaged at the external rim of the ear.

A total of 5,000 cells were plated in 96-well plates for 24 hours. Bromodeoxyuridine or 5-bromo-2′-deoxyuridine (BrdU) (Calbiochem, Billerica, MA, USA) assay was carried out according to the manufacturer’s instructions. Absorbance at 450 nm was read using a Tecan Infinite plate reader. In parallel, 150,000 cells were plated in 24-well plates for 24 hours. Cells were incubated with BrdU label (1:2000) for 20 hours, treated with a fixative/denaturing solution (30 minutes) and incubated with an anti-BrdU antibody (1:1000) and rat anti-mouse BST-2 antibody (1:200, eBioscience) for 1 hour at room temperature. Cells were washed and incubated with Alexa Fluor 594 anti-rat (Invitrogen, Waltham, MA, USA) and Alexa Fluor 488 anti-mouse (Invitrogen) secondary antibodies for 30 minutes at room temperature. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI)-containing Vectashield (Vector Laboratories, Burlingame, CA, USA) and imaged using a Zeiss 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). Images were processed using Image J software. BrdU label, fixative/denaturing solution, and anti-BrdU antibody were from BrdU (Calbiochem) assay.