Rat anti meca79

KLNs were frozen and sectioned with a cryomicrotome. After blocking with 3% (vol/vol) bovine serum albumin in phosphate-buffered saline (PBS) for nonspecific antigens, sections were incubated overnight with primary antibodies at 4°C, followed by secondary conjugated antibodies for 1 hour at room temperature. The following antibodies were used: rat anti-MECA79 (Novus Biologicals, 1:200), goat anti-PDPN (R&D Systems, 1:200), rabbit anti-fibronectin (Abcam, 1:300), rabbit anti-collagen I (Abcam,1:300), rabbit anti-LYVE-1 (Abcam, 1:300), fluorescein (FITC)-conjugated rat anti-LYVE-1 (BioLegend, 1:100), and rat anti-TGFβR1 (Santa Cruz, 1:200). The secondary antibodies used were either FITC-anti-rat, Cy3-anti-rabbit/goat, or aminomethylcoumarin acetate (AMCA)-anti-goat (Jackson ImmunoResearch, 1:200). Images were captured using a EvosFL Auto2 fluorescence microscope. Areas of positive staining were assessed semi-quantitatively by ImageJ software (NIH). Statistical analysis was performed using two-tailed Student’s t tests by Prism (GraphPad Software, Inc).

PLNs were embedded in optimum cutting temperature compound and stored at −80 °C. Then, the frozen sections were cut at 8μm and washed with PBS for 5 min. Samples were blocked with 3% (vol/vol) bovine serum albumin in PBS and incubated with primary antibodies overnight. Sections were washed 3 times with PBS and incubated with secondary conjugated antibodies at room temperature. The following antibodies were used for the staining: goat anti-PDPN (R&D Systems, 1:200), rat anti-Meca79 (Novus Biologicals, 1:200), rabbit anti-Fibronectin (Abcam, 1:300), rat anti-ERTR7 (Santa Cruz Biotechnology, 1:100), rabbit anti-collagen IV (Abcam,1:300), rat anti-B220 (Invitrogen, 1:200), rabbit anti-CD3 (Abcam, 1:250), rabbit anti-lyve-1 (Abcam, 1:300). Secondary antibodies were either FITC- or Cy3-conjugated (Jackson ImmunoResearch, 1:200). DAPI (VECTASHILED, Vector Laboratories) mixed in Prolong-Gold mounting media was used as a nuclear counterstain. Images were obtained by EvosFL Auto2 microscopy. All images were automatically processed using ImageJ (NIH) and split into RGB channels. Auto threshold was used to convert intensity values of the immunofluorescent staining into numeric data.