Rabbit anti fibronectin

Tissues were collected at the designated times and snap-frozen in optimal cutting temperature (OCT) medium (Sakura Finetek, Japan), and 8-μm sections were prepared and stained with the following antibodies; Rabbit anti-fibronectin (1:100) (Millipore, Temecula, CA, USA), rabbit anti-vimentin (1:100) (Cell Signalling Technology, Danvers, MA, USA), rabbit anti-N-cadherin (1:1000) (Thermo Scientific, Rockford, IL, USA), as described previously^{13 (link),41 (link)}. Stained sections were analysed using fluorescent or bright-field imaging microscopy (Leica, Germany) and ImagePro Plus Capture and Analysis software (Media Cybernetics, Rockville, MD). Vimentin, Fibronectin and N-Cadherin positive areas were quantified in 15 independent fields/section using Image Pro-Plus software^{41 (link),44 (link)}.

Frozen sections of 12 μm thickness were subject to a standard protocol for immunofluorescence staining as described previously (Zhao et al., 2008 (link)). Where required, heat-mediated antigen retrieval was performed using a commercial antigen retrieval solution (Sigma-Aldrich). The following antibodies were used: goat /rabbit anti-GFP (Abcam), rabbit anti-Olig2 (Millipore), rabbit anti-GFAP (Dako), rabbit anti-periaxin (gift from Professor Peter Brophy or from Sigma-Aldrich), rabbit anti-S100β (Dako), rat anti-PDGFRa (CD140a; BD Bioscience), rabbit anti-prolyl-4 hydroxylase β (P4HB; Abcam), rabbit anti-HSP47 (BioVision), rabbit anti-IBA1 (Wako), rabbit anti-smooth muscle actin (SMA; Abcam), rabbit anti-Ki67 (Abcam), chicken anti-myelin protein zero (P0) (Abcam), goat anti-Sox2 and goat anti-Sox10 (Santa Cruz Biotechnology), rat anti-CD31 (BD Biosciences), rabbit anti-fibronectin (Millipore), rat anti-L1cam (Millipore), and rabbit anti-Foxj1 (Insight Biotechnology) Secondary antibodies against relevant primary antibodies labeled with either Alexa Fluor 488 or Alexa Fluor 594 were from Thermo Fisher Scientific. The images were acquired with a Leica SP5 confocal microscope or a Zeiss Axio Observer A1 fluorescence Imaging System.

Tumour and lung tissues were harvested and snap-frozen in optimal cutting temperature (OCT) medium (Sakura Finetek, Torrance, CA, USA), and 8-μm sections were prepared. Tumour sections were stained with the following antibodies: rabbit anti-fibronectin (Millipore, Temecula, CA, USA), rabbit anti-vimentin (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-N-cadherin (Thermo Scientific, Rockford, IL, USA), followed by Alexa 488 or Alexa 568-conjugated goat anti-rabbit IgG. Lung sections were stained with hematoxylin (Vector Laboratories, Burlingame, CA, USA) and eosin (Fisher Scientific Co., Kalamazoo, MI, USA) following the manufacturer’s protocol. We used fluorescent or bright-field imaging microscopy (Leica, Wetzlar, Germany) and ImagePro Plus Capture and Analysis software (Media Cybernetics, Rockville, MD, USA) to analyse the stained sections. We used Image Pro-Plus software to quantify stained areas from 10–15 independent fields/sections.

Cells grown on coverslips were fixed with 3% paraformaldehyde and stained with primary and fluorescently labeled secondary antibodies. The following antibodies were used: mouse anti-α-smooth muscle actin (α-SMA) (clone 1A4, Sigma, Darmstadt, Germany), mouse anti-synaptopodin (clone D-9, Santa Cruz, Santa Cruz, CA, USA), mouse anti-cytokeratin 18 (CK18) (clone RGE-53, Merck Millipore, Darmstadt, Germany), rabbit anti-fibronectin (Sigma), mouse anti-integrin α_vβ₃ (clone LM609, Millipore), mouse anti-CD31 (Dako, Glostrup, Denmark), and mouse anti-N protein PUUV (A1C5, Progen, Heidelberg, Germany). Cell nuclei were stained by Hoechst 33342 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Images were taken using an Axiocam 506 mono camera attached to an Axio Observer.D1 inverted microscope (Carl Zeiss, Oberkochen, Germany). For Western blot analysis, the following primary antibodies were used: rabbit anti-PUUV N protein and mouse anti-α-tubulin (Sigma). Loading was verified by the detection of tubulin on the same membrane. Detection was performed by using near infrared fluorescent dye (IRDye)-conjugated secondary antibodies and an Odyssey CLx infrared imaging system (Li-Cor, Lincoln, NE, USA).

Sections were deparaffinized and rehydrated as described above. For immunostainings of Wilms tumor protein (WT1), cleaved caspase-3 and collagen I, antigen retrieval was performed using Tris/EDTA buffer (pH 9.0, 10 mM; 20 min, 100 °C), citrate buffer (pH 6.0, 10 mM; 20 min, 100 °C) or pepsin (0.4% in 0.1 HCL; 15 min, 37 °C), respectively. Endogenous peroxidase was blocked with 0.12% H₂O₂ in Milli-Q (20 min, RT), followed by incubation with primary antibody diluted in 1% BSA in PBS. Antibodies used were (1) rabbit anti-fibronectin (Sigma-Aldrich, St.Louis, MO); (2) rabbit anti-collagen I (AbD Serotec, Puchheim, Germany); (3) rabbit anti-WT1 (Santa Cruz Biotechnology, Santa Cruz, CA); and (4) rabbit anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA). Negative controls for immunohistochemistry included normal sera of the same species as the primary antibody. The immunoreactions were visualized with 3,3-diaminobenzidine (Dako), counterstained with hematoxylin, dehydrated, and mounted. Cell numbers were determined by randomly analyzing 25 glomeruli of each experimental animal. Sections stained for fibronectin and collagen I were digitalized and graded blindly on a scale of I to V depending on the staining intensity: I, none; II, minute; III, moderate; IV, high; V, very high.