Cy3 labeled goat anti rabbit igg

Mice femurs, and spleens were isolated and fixed in 4% PFA (Sigma) for 48h, and subsequently embedded in Optimal Cutting Temperature (O.C.T) (Trajan, Australia), sectioned at 10μm, and Immunofluorescence stained. Before embedding, femurs needed to be further decalcified in 5% nitric acid for 48 hours. Spleen and femurs sections were blocked with 1% BSA and 5% goat serum in PBST (0.1% tween-20 in PBS) for 1 hour and incubated with rabbit anti mouse CD61 (Abcam) overnight. After incubation, cells were stained with Cy3-labeled goat anti–rabbit IgG (Jackson ImmunoResearch, USA) for 1hr, followed by 5 min staining of 4, 6-diamidino- 2-phenylindole (DAPI, Sigma-Aldrich).
Tetracycline (tet)-on inducible control and HGPS K562 cells were harvested, followed by fixation with 4% PFA and permeabilization by 0.5% Triton X. Fixed cells were incubated with anti-LaminA, anti-LPA₃ (ab108595 and ab23692; Abcam) and anti-GFP (sc-9996; Santa Cruz) primary antibodies overnight and then labeled with Alexa Fluor 647 and FITC fluorescent secondary antibody (A-31573 and A16018; Invitrogen, Carlsbad, CA, USA) for 1 hr. All slides were mounted with Fluoromount-GTM (Emsdiasum, Fort Washington, PA, USA) and imaged by Zeiss AxioPlan 2 fluorescence microscope (Zeiss, Jena, Germany).

Spleens were harvested from the mice after NP-Ficoll immunization at indicated times, snap-frozen in liquid nitrogen and frozen in Tissue-Tek OCT compound (Sakura) at −80°C. For immuno-fluorescence of IDO1 5μm frozen splenic sections were immediately fixed in −20°C methanol. The sections were blocked with PBS containing 1% nonfat milk (Sigma) and stained with 4μg/mL polyclonal rabbit anti-mouse IDO1 (a gift from Dr. David Munn) in PBS containing 1% nonfat milk. After extensive washing with TBS + 0.05% tween 20 (ACROS Organics), the sections were stained with a 1:400 dilution of CY3-labeled goat anti-rabbit IgG (Jackson Immunoresearch), APC-labeled anti-B220 (Ebioscience), or FITC-labeled anti-CD138 (BD Pharmingen). To examine antibody response to NP-Ficoll immunization, sections were incubated with anti-IgM (BioLegend) and anti-IgG3 (Southern Biotech) in blocking buffer for 60 minutes at room temperature in the dark. For stimulated and unstimulated purified B cells, cytospins were prepared and fixed with −20°C methanol. Cells were made permeable using Perm Buffer III (BD Biosciences) and stained for IDO1 as explained above and anti-B220 (eBioscience). Sections and cytospins were mounted with Prolong Gold anti-fade with DAPI (Invitrogen). Fluorescent images were captured using a Zeiss LSM 510 Meta confocal microscope equipped with 405-, 488-, 561-, and 633-nm lasers.