Fitc conjugated antibodies

Monoclonal antibodies (mAb) 1A8 (specific to the HTNV NP; Xu et al., 2002 (link)), Gn-4 (specific to the HTNV Gn), Gc-10 (specific to the HTNV Gc), and 3G1 (with high neutralizing Ab activity against HTNV) were prepared in our laboratory. Our laboratory also supplied the mAb Sp2/0 (Cheng et al., 2014 (link); negative control). Rat anti-mouse GM-CSF and rabbit anti-mouse CD40L antibodies were purchased from Abcam (Cambridge, MA, USA). Other secondary antibodies, including FITC-conjugated antibodies, Cy3-conjugated antibodies, HRP-conjugated antibodies, and PE-conjugated antibodies were purchased from eBioscience (San Diego, CA, USA).

To determine the surface level of AT1R, vascular cell adhesion molecule 1 (VCAM-1), endothelial-selectin (E-selectin) and platelet-selectin (P-selectin) on HUVECs and REAC, cells were incubated with FITC-conjugated antibodies (eBioscience). Stained cells were analyzed on a FACSCalibur using CELLQuest software (Becton Dickinson).

Microdissection was performed using an Arcturus XT microdissection system (Applied Biosystems). Eight-μm frozen tissue sections were cut and mounted on membrane-coated glass slides (LCM522, Applied Biosystems). Slides were stained in 1% methylene green (diluted in DEPC-treated water) for 10 seconds, washed three times in DEPC-treated water, and used immediately for microdissection. For RNA extraction, captured samples were collected in TRI Reagent (Sigma), and RNA extraction was performed using a standard protocol. For immunofluorescence-guided LCM, frozen blocks of normal skin and in situ SCCs were cut and fixed briefly with 75% ethanol for 30 seconds. After a brief blocking procedure (in 5% bovine serum albumin (BSA) (SIGMA) in nuclease-free PBS (GIBCO, Thermo Fisher) for 2 min), sections were incubated with a mixture of FITC-conjugated antibodies (dilution 1:1000, ThermoFisher) against PDGFRα and propidium iodide (SIGMA, P4170) for 2 min, followed by quick rinsing with PBS. The air-dried sections were then used to fluorescence-guided LCM using an Arcturus XT microdissection system as before. According to the manufacturer’s recommendations, the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) was used for RNA extraction.