Mouse anti cd34

Immunohistochemistry and immunofluorescence were performed as described in our previous study [19 (link), 20 (link)]. Primary antibodies were rabbit anti-AGGF1 (ab203680, 1:200), mouse anti-VEGF (ab1316, 1:150) and mouse anti-CD34 (ab8536, 1:400, all from Abcam, USA). The HRP-conjugated second antibody was from Invitrogen, Carlsbad, CA and the diaminobenzidine was from Beijing Zhongshan Golden Bridge Biotech, China. As for immunofluorescence, the primary antibodies cocktail and the mixture of secondary antibodies were rabbit anti-AGGF1 (1:150), mouse anti-CD34 (1:200) and Alexa Fluor 488-conjugated donkey anti-rabbit, Alexa Fluor 568 donkey anti-goat (all from Invitrogen), respectively.
For the negative control, the primary antibody was carried out with phosphate-buffered saline (PBS).

Dual IF was used to visualize protein colocalization. As described in the IHC protocol, adjacent slides were deparaffinized, followed by antigen retrieval in sodium citrate solution for 15 min at 98 °C, and then blocked for 30 min using Bloxall (Vector Labs). Primary antibodies were added, and the slides were incubated at 4 °C overnight. The primary antibodies used are as follows: rat anti-LANA (Abcam, 1:100), mouse-anti CD34 (Invitrogen, 1:200), rabbit-anti Prox-1 (Abcam, 1:500), rabbit-anti KDR (ProteinTech, 1:200), rabbit-anti FLT4 (Invitrogen, 1:100), rabbit-anti ADAM12 (Invitrogen, 1:100), rabbit-anti UNC5A (ProteinTech, 1:100), rabbit-anti OX40 (Novus, 1:100), and rabbit-anti ZP2 (Invitrogen, 1:100). After washing, the slides were incubated with their corresponding secondary antibody (chicken anti-rat AF647 (Invitrogen, 1:100), donkey anti-mouse AF488 (Invitrogen, 1:100), donkey anti-rabbit AF488 (Invitrogen, 1:100), donkey anti-rabbit AF647 (Invitrogen, 1:100)) for 2 h at RT. The slides were then counterstained with 300 nM DAPI for 30 min at RT and mounted using Fluoro-gel (Electron Microscopy Sciences, Hatfield, PA, USA).

Formalin-fixed, paraffin-embedded (FFPE), KS tumor biopsy specimens were sectioned into adjacent 6 μm-thick sections for protein detection, as described in Tso et al. [22 (link)]. Briefly, the slides were deparaffinized in xylene and rehydrated in graded ethanol washes, followed by 30 min treatment with 2% hydrogen peroxide in methanol to quench endogenous peroxidase activity. The slides then underwent antigen retrieval in sodium citrate solution pH 6.0 for 15 min at 98 °C. The slides were blocked for 30 min using Bloxall (Vector Labs, Newark, CA, USA), followed by primary antibody application and overnight incubation at 4 °C. The antibodies used for IHC were as follows: mouse anti-LANA (Leica, Deer Park, IL, USA, 1:100), rat anti-LANA (Abcam, Boston, MA, USA, 1:100), mouse-anti CD34 (Invitrogen, Boston, MA, USA 1:200), rabbit-anti Prox-1 (Abcam, 1:500), rabbit-anti KDR (ProteinTech, Rosemont, IL, USA, 1:1500), rabbit-anti FLT4 (Invitrogen, 1:800), rabbit-anti ADAM12 (Invitrogen, 1:500), rabbit-anti UNC5A (ProteinTech, 1:300), rabbit-anti OX40 (Novus, Centennial, CO, USA 1:500), rabbit-anti ZP2 (Invitrogen, 1:5000). The slides were then washed, and the species-appropriate HRP-labeled polymeric secondary reagent was applied for 30 min at RT, followed by the addition of diaminobenzidine for chromogen deposition. Hematoxylin was used for counterstaining.