Alexa fluor 647 conjugated anti rabbit igg

Fluorescent IHC staining was conducted for FFPE synovial tissues collected from RA and OA specimens, as previously reported [24 (link)]. The following primary antibodies were used: anti-NRP1 antibody (rabbit monoclonal, clone EPR3113; Abcam, Cambridge, UK), anti-NRP2 antibody (rabbit monoclonal, clone D39A5; Cell Signaling Technology, Tokyo, Japan), anti-human cadherin-11 (CDH11) antibody (goat polyclonal; R&D Systems Inc.), anti-ACE2 antibody (rabbit polyclonal; Bioss Antibodies Inc., Woburn, MA, USA), and anti-TMPRSS2 antibody (rabbit polyclonal; Proteintech, Tokyo, Japan). The following secondary antibodies were used: Alexa Fluor 405-conjugated anti-goat immunoglobulin G (IgG) (host: donkey; Abcam) and Alexa Fluor 647-conjugated anti-rabbit IgG (host: donkey; Abcam). The slides were quenched using the Vector TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, Burlingame, CA, USA). Nuclear staining was performed with acridine orange (Abcam). Subsequently, glass slides were treated with VECTASHIELD Vibrance Antifade Mounting Medium (Vector Laboratories). Fluorescent images were obtained using a digital microscope VHX-7000 (KEYENCE, Osaka, Japan).

Paraffin-embedded and formalin-fixed HCC samples were used for immunohistochemistry detection as previously described55 (link). Antibodies (Ab) including human EGFR (1:100, ab30), Foxp3 (1:100, ab2481), IFN-γ (1:200, ab9657), cleaved Caspase 3 (C.C3) (1:200, ab2302) and IL-10 (1:100, ab34843) (Abcam, Cambridge, UK) were used. For immunofluorescence analysis, tissue slides were stained with rabbit anti-human Foxp3, rabbit anti-human cleaved Caspase 3, mouse anti-human EGFR, and goat anti-human IFN-γ antibodies, followed by staining with Alexa Fluor 488-conjugated anti-mouse IgG (1:500, Ab150117), Alexa Fluor 555-conjugated anti-rabbit IgG(1:1,000, Ab150074), and Alexa Fluor 647-conjugated anti-Rabbit IgG (1:500, Ab150075) (Abcam, Cambridge, UK) antibodies. Positive cells were quantified using Image-Pro Plus software (Media Cybernetics, MD, USA) and detected by confocal microscopy (Zeiss, Oberkochen, Germany).