2 k ccd camera

For TEM studies, samples were embedded in LR White resin (Merck KGaA, Darmstadt, Germany) and cut to a thickness of 70 nm on a Reichert-Jung UltraCut E microtome. Immunogold staining was performed post embedding using the polyclonal primary anti-GFAP antibody (DAKO) 1:200 in MSB for 2 h at room temperature. A colloidal gold 18 nm secondary antibody was used diluted 1:50 in MSB with incubation time of 2 h at room temperature. Subsequently, samples were post fixed in 1% GA and incubated in 1% aqueous Uranyl acetate and 1% lead nitrate respectively for an overall enhanced tissue contrast. Imaging was performed on a transmission electron microscope Zeiss EM 902A equipped with a wide-angle dual speed 2 K-CCD-Camera at 80 kV acceleration voltage.

TEM samples after fixation were treated and stained as previously described by Davalli and colleagues [21 (link)] and by Reynolds [22 (link)]. Samples were further analyzed using a transmission electron microscope (EM 10A, Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with a 2K‐CCD‐Camera (TRS). For each mouse, 5 peripheral tumor associated lymphatics were chosen and serial ultrathin sections were analyzed in order to quantify the frequency of the canalicular formation in each lymphatic and how many times a TCi or a leukocyte was found within the formed channel. The normality distribution of the data was assessed with the Shapiro-Wilk test and the unpaired t test was further applied. Statistical significance for each analysis was set at a p-value ≤ 0.05. All statistical analyses were performed with GraphPad Prism version 8.0.1 for Windows (GraphPad Software, La Jolla, CA, USA).