Texas red x goat anti rat igg

Frozen 5 μm-thick tissue sections were fixed in cold acetone for 15 min, air-dried, and treated for 30 min with a blocking reagent [10% fetal bovine serum (FBS) in PBS]. After washing with PBS, the sections were incubated with primary antibodies overnight at 4°C, and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) for anti-NP and anti-pSFK (Tyr416) antibodies, Alexa Fluor 594 donkey anti-goat IgG (Invitrogen) for anti-MGL1/2 antibody, Texas Red-X goat anti-rat IgG (Invitrogen) for anti-CD3 antibody, and Alexa Fluor 594 donkey anti-rabbit IgG (Invitrogen) for anti-MPO antibody for 2 hrs at room temperature (RT). The sections were mounted with CC/Mount (Diagnostic BioSystems, Pleasanton, CA) containing DAPI (Dojindo Laboratories, Kumamoto, Japan). Fluorescent images were visualized using BIOREVO BZ-9000 (Keyence, Osaka, Japan).

Five micrometer-thick tissue sections on glass slides (Matsunami, Tokyo, Japan) coated with poly-L-lysin (Wako Pure Chemicals, Osaka, Japan) were deparaffinized and rehydrated, and treated with proteinase K (Wako Pure Chemicals, 20 mg/ml in 10 mM Tris HCl, pH 7.6) at 37°C for 30 min. After washed with PBS, the sections were incubated with primary antibodies against PrP (IBL-N, Immuno Biological Laboratories, Gunma, Japan), podoplanin (MBL, Nagoya, Japan), SP-C (Santa Cruz Biotechnology), CC10 (Santa Cruz Biotechnology), and NP virus protein (GeneTex) overnight at 4°C, and stained with Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen) for IBL-N anti-PrP antibodies, Texas Red-X goat anti-rat IgG (Invitrogen) for anti-podoplanin antibody, Alexa Fluor 488 donkey anti-goat IgG (Invitrogen) for anti-SP-C and anti-CC10 antibody, and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) for anti-NP antibody for 2 h at room temperature. The sections were mounted with CC/Mount (Diagnostic BioSystems, Pleasanton, CA) containing DAPI (Dojindo Laboratories, Kumamoto, Japan). Fluorescent images were visualized using BIOREVO BZ-9000 (Keyence, Osaka, Japan).