Alexafluor 488 conjugated goat anti rabbit antibodies

5–10 μm cryosections of patient and control biopsies were stored until analysis at −80 °C. Sections were thawed before staining. Primary antibodies were directed against CD68, clone KP1 (M0814; DAKO; 1:800), eMyHC (F1.652; DSHB; 1:150), Ki67 (Ab15580; Abcam; 1:50), Laminin (L9393; Sigma; 1:500 or LS-C96142; LS Bio; 1:500), MyoD (SC304; Santa Cruz; 1:200), Myogenin (M-225 ; Santa Cruz; 1:200), Pax7 (DSHB; 1:50), CD3 (2GV6; Ventana; ready to use), CD20Cy; clone L26; Dako, 1:400). Following the primary antibody incubation, sections were rinsed and incubated with biotin-conjugated anti-mouse antibody (BA-2000; Vector labs,1:50) and then with Alexafluor594-conjugated streptavidin (S11227; Life Technologies, 1:500). Sections incubated with rabbit primary antibodies were subsequently incubated with Alexafluor 488-conjugated goat-anti rabbit antibodies (A11307; Life Technologies,1:500). Finally chicken anti-Laminin was detected with Alexafluor647-conjugated goat anti-chicken antibodies (A21449; Life Technologies, 1:500). All sections were counterstained with Hoechst33258 (H3569; Life Technologies, 1:15000). The slides were mounted with Mowiol (475904; Calbiochem). For some biopsies, limited amounts of sections of sufficient quality were available for immunofluorescent analysis, and not all stainings could be performed for all patients.

Six-micron frozen DLE (n = 6–8) and normal (n = 4–5) skin sections fixed in acetone were blocked in 5 % normal goat serum in PBS with 0.3 % Triton X-100 for 1 hour. Slides were then incubated with rabbit anti-human primary antibodies—CD127 (Abcam, Cambridge, MA, USA), CD209 (Abcam), CXCL10 (PeproTech, Rocky Hill, NJ, USA), tumor necrosis factor-α (TNF-α) (Novus, Littleton, CO, USA), and TGF-β (Novus)—overnight followed by PBS washes. Alexa Fluor^® 488-conjugated goat anti-rabbit antibodies (Life Technologies, Carlsbad, CA, USA) were added for 30 minutes followed by PBS washes, except for TGF-β and TNF-α antibody-stained slides, whose signals were amplified by using the TSA Biotin kit in accordance with the instructions of the manufacturer (PerkinElmer, Waltham, MA, USA). Mouse anti-human CD163 antibody (Novus) or mouse anti-human CD3 antibody (eBioscience, Inc., San Diego, CA, USA) was added for 1 hour, followed by PBS washes. Slides were incubated with Alexa Fluor^® 594-conjugated goat anti-mouse antibodies (Life Technologies) for 30 minutes and washed with PBS. Slides were coverslipped with Vectashield mounting medium and analyzed. Images were acquired with an Olympus BX60 fluorescent microscope (Olympus, Tokyo, Japan).