Strepta vidin dylight 550

For immunofluorescence staining of tissues, 10 μm cryosections were equilibrated at RT, fixed in PFA for 15 min, permeabilized using PBS containing 0.2% Triton-X for 10 min, and blocked in PBS containing 0.05% Tween-20, 5% FBS, 5% BSA, and 5% goat serum (GE Healthcare, UK) for 1 h. The sections were immunostained with rabbit anti-fluorescein (cat. no. A889, Thermo Fisher Scientific, MA, USA), rat anti-mouse CD31, biotin rat anti-mouse CD11b (cat. no. 553370; 557395, BD Biosciences, CA, USA), rat anti-mouse LYVE-1 (cat. no. 14044382, eBioscience, CA, USA), rabbit polyclonal anti-Ki67 (cat. no. NB500–170, Novusbio, UK), and rabbit anti-Cleaved Caspase-3 (Asp 175),(cat. no. 966, Cell Signaling Technology, Inc., MA, USA) as primary antibodies,. Alexa 488-conjugated goat anti-rabbit IgG, Alexa 647-conjugated goat anti-rat IgG, Alexa 546-conjugated goat anti-rabbit IgG (all Invitrogen, Thermo Fisher Scientific, MA, USA) and strepta- vidin Dylight^® 550 (Thermo Fisher Scientific, MA, USA) were used as secondary antibodies. Nuclei were counterstained with 10 pg/ml DAPI. The stained tissue sections were examined by fluorescence confocal microscopy using Olympus FV1200MPE instrument, and the images were processed and analyzed using the FV10-ASW 4.2 Viewer image software (Olympus, Germany) and Image J freeware.

Frozen 0.7μm sections were fixed in acetone:ethanol, blocked sequentially with anti-Fc (2.4G2) (BioXcell), Avidin/Biotin Blocking Kit (Vector), H₂O₂ and NaN₃, then stained with either CD31-FITC (eBioscience) or CD31-AF647 (Biolegend), and MadCAM-1-Biotin, VCAM-1-Biotin, or ICAM-1-Biotin (all eBioscience). Streptavidin DyLight550 (ThermoFisher) was used as secondary. Perkin Elmer TSA Biotin Kit was used for amplification. Images were collected on an AxioImager with Apotome (Zeiss). ImageJ software (NIH) was used to quantify CD31⁺ pixels that were also VCAM-1⁺, MadCAM-1⁺, HA⁺, or ICAM-1⁺ (32 (link)).