Microphot fxa fluorescence microscope

Frozen sections on microscopy slides were fixed in 4% paraformaldehyde for 20 min, followed by an incubation with 0.1 M glycine for 20 min and blocking in 10% non-immune serum of the secondary antibody species in PBS containing 0.1% Triton X-100 for 30 min. Slides were then incubated with the primary antibodies in 1% BSA/PBS for 2 h at room temperature, as it follows:
1:200 FITC-labeled mouse anti-rat a-smooth muscle actin (a-SMA, Sigma-Aldrich, St Louis, MO), 1:100 rabbit anti-rat F4/80 (Abcam, Cambridge, MA) and 1:100 rabbit anti-rat myeloperoxidase (MPO, eBioscience, San Diego, CA). Sections were then washed three times with 0.1% BSA in PBS and in the case of MPO incubated with 1:25 FITC-labeled goat anti-rabbit secondary antibody (Life Technologies, Grand Island, NY), for 1 h at room temperature. After washing, slides were mounted in Vectashield containing DAPI (Vector Laboratories, Inc. Berlingame, CA). Immunofluorescence was visualized and imaged using a Nikon Microphot-FXA fluorescence microscope with a Nikon DS-Fi1 digital camera and NIS-Elements software (Nikon Instruments Inc., Melville, NY).

Frozen sections were fixed in 4 % paraformaldehyde for 20 min, rinsed with PBS, incubated in a solution of 0.1 M glycine in PBS for 20 min and blocked in 10 % non-immune serum of the secondary antibody species in PBS containing 0.1 % Triton X-100 for 30 min. Slides were then incubated for 2 h at room temperature with the primary antibodies in 1 % BSA/PBS. The primary antibodies used, and respective dilutions, were the following: 1:200 FITC-labelled mouse anti-mouse α-smooth muscle actin (αSMA, Sigma-Aldrich) and 1:100 rabbit anti-mouse fibrinogen (Abcam). Sections were then washed three times with 0.1 % BSA in PBS and incubated with 1:25 goat anti-rabbit secondary antibodies (Life Technologies), for 1 h at room temperature. After washing, sections were mounted in Vectashield containing DAPI (Vector Laboratories). Immunofluorescence was visualized and imaged using a Nikon Microphot-FXA fluorescence microscope with a Nikon DS-Fi1 digital camera and Nikon NIS-Elements software (Nikon Instruments).