Fluorescein isothiocyanate conjugated goat anti rabbit igg

Indirect immunofluorescence staining was performed to detect the expression of collagen I and collagen III in the paraffin sections of LAA tissues. Tissue sections were blocked with normal bovine serum albumin (Zhongshan Goldenbridge Biotechnology, China) for 30 min, and then, the sections were incubated with polyclonal rabbit anti-human collagen I antibody (1 : 50, ab34710, Abcam) or monoclonal mouse anti-human collagen III antibody (1 : 200, ab6310, Abcam) overnight at 4°C. After washing with PBS solution with 0.1% polysorbate, the sections were incubated with the corresponding secondary antibodies at 37°C for 1 h. Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1 : 100, Zhongshan Goldenbridge Biotechnology) was used as the secondary antibody for collagen I, and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1 : 100, Zhongshan Goldenbridge Biotechnology) was used as the secondary antibody for collagen III. Finally, the sections were washed with PBS solution thrice and counterstained with 4′,6-diamidino-2-phenylindole (1 : 100) for 1 h at room temperature. Negative controls were conducted without incubation with primary antibodies.

Immunofluorescence staining was performed on the frozen sections of the FVMs by staining with the following antibodies: rabbit anti-EPO polyclonal IgG (1:150 dilution; No. ab126876 Abcam, Cambridge, MA, USA), mouse anti-CD105 monoclonal IgG (1:150 dilution; No. ab69772 Abcam, Cambridge, MA, USA), rabbit anti-VEGF polyclonal IgG (1:200 dilution; No. ab39250 Abcam, Cambridge, MA, USA), tetramethylrhodamine isothiocyanate- conjugated goat anti-mouse IgG (1: 200 dilution; Zhongshan Goldenbridge Biotechnology Co. Ltd., Beijing, China), and/or fluorescein isothiocyanate- conjugated goat anti-rabbit IgG (1: 200 dilution; Zhongshan Goldenbridge Biotechnology Co. Ltd.). The samples were counterstained with 4′,6′-diamino- 2-phenylindole (DAPI) (1: 1,000 dilution, No. D9542; Sigma-Aldrich, St. Louis, MO, USA). All the sections were examined using a fluorescence microscope (DS-Ril-U2; Nikon, Tokyo, Japan) and photographed (DS-U2; Nikon).

Frozen sections were fixed in an acetone solution for 30 min, incubated with 0.4% Triton for 30 min, and then incubated with 3% normal goat serum at room temperature for 1 h. Between every step, the sections were washed with PBS. For double staining, the sections were incubated with anti-RASgrf1 (1:30; Santa Cruz, USA), anti-microtubule-associated protein 2 (MAP2) (1:100; Boster, China) and anti-glial fibrillary acidic protein (GFAP) (1:100; Boster, China) at 4°C overnight. Next, the sections were washed and incubated in fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:50; Zhongshan Golden Bridge, China) and tetramethyl rhodamine isothiocyanate-conjugated goat anti-mouse IgG (1:50; Zhongshan Golden Bridge, China) in the dark for 60 min at room temperature. Fluorescent images were collected by laser scanning confocal microscopy (Leica, Germany) using an Olympus IX 70 inverted microscope (Olympus, Japan).