Alexa fluor 546 conjugated goat anti rat igg

Frozen-fixed LV sections (5 μm) were incubated with primary rat monoclonal antibodies against mouse CD45 (1:50, BD Pharmingen, USA) or mouse CD68 (1:200, AbD Serotec, USA), followed by incubation with secondary antibody Alexa Fluor 546-conjugated goat anti-rat IgG (1:500, Invitrogen, USA), as previously described^{35 (link)}. Nuclei were stained with ProLong® Gold antifade reagent with 4′, 6-diamidino-2-phenylindole (Invitrogen). LV sections (8∼10 per heart) were acquired using Olympus BX61 fluorescence microscope and AnalySIS FIVE software (Olympus, Australia) at x20 magnification. The number of inflammatory cells was manually quantified, in a blinded fashion, using ImageJ (NIH, USA) and the average value was used.

The liver from C57BL/6 mice were dissected and fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4) at 4°C, overnight. The tissue was then cryoprotected with series of sucrose gradient (10%, 20% and 30% w/w) in phosphate buffer at 4°C and cut into 20-μm sections. The sections were washed 3 times in phosphate-buffered saline (PBS), blocked in PBS containing 5% goat serum, 1 mg/mL bovine serum albumin (BSA) and 0.1% Triton X-100 for 1 hour at room temperature and incubated with monoclonal rat anti-CXCL1 (1:100; clone: 124014, R&D), polyclonal rabbit anti-CRBP1 (1:50; FL-135, Santa Cruz) or Alexa Fluor 647-conjugated rat anti-F4/80 antibody (Serotec) in PBS containing 1mg/mL BSA and 0.1% Triton X-100, at 4°C, overnight. The sections were washed and stained with Alexa Fluor 546-conjugated goat anti-rat IgG (for CXCL1) or Alexa Fluor 488-conjugated goat anti-rabbit IgG (for CRBP1) (1:500; Invitrogen). In genetically modified mice, GFP expression under Tie-2 promoter restricted GFP signal to LSECs. Sections were stained with TO-PRO-3 (1:1000; Invitrogen) for nuclear visualization in HSCs. All sections were mounted with SlowFade Gold antifade reagent (Invitrogen) and observed with a Leica SL confocal microscope under a 100X objective lens (Leica Microsystems) located in the Keck Imaging Facility at the University of Washington.