Mouse anti human vimentin

Cryopreserved sections were blocked with 5% BSA/5% horse serum (Sigma) and incubated overnight with primary antibodies: rabbit anti-human K5 (1:500, Biolegend), mouse anti-human K10 (1:500, DAKO), mouse anti-human Col IV (1:500, Sigma), rabbit anti-human CD29 (1:200, GeneTex, Irvine, USA), mouse anti-human Ki67 (1:100, DAKO), mouse anti-human Vimentin (1:200, DAKO), rabbit anti-human pan cytokeratin (1:100, Novus Biological, Littleton, USA) or mouse anti-human α5 chain Laminin (1:500, Millipore, Burlington, USA). Slides were washed and incubated with Alexa Fluor-conjugated mouse or rabbit secondary antibodies (BD Biosciences, Columbus, USA). Sections were imaged on a Nikon Ti-E microscope. Ki67 staining was quantitated for fluorescent nuclei with the ‘Tissue Analysis Cell Counter’ macro using FIJI software (NIH). Ki67 and DAPI nuclear signal thresholds were adjusted to minimise nuclear segmentation or fusion and verified by manual count.

For immunofluorescence studies, specimens were fixed for 20–25 min in 4% paraformaldehyde (PFA), rinsed in PBS, transferred into 10% and 30% sucrose in PBS, and embedded in tissue freeze medium (Tissue Tek, Sakura Finetek Zoeterwoude, NL). Sections of 12–14μm were incubated with the following primary antibodies: Rabbit-anti-human CCBE1 (1:500, Sigma-Aldrich, München, Germany), rabbit-anti-human ESAM-1 (1:100, Sigma-Aldrich, München, Germany), goat-anti-human smooth muscle α-actin (SMA) (Acris Antibodies, Herford, Germany), mouse-anti-human CD31 (1:50, BD Pharmingen), mouse-anti-human CD117 (c-KIT, 1:100, Santa Cruz Biotechnol., and 1:1, Dako, Hamburg, Germany), mouse-anti-human D2-40 (1:200, Dako, Hamburg, Germany), rabbit-anti-human Lyve-1 (1:500, ReliaTech, Braunschweig, Germany), rabbit-anti-human Prox1 (1:500, ReliaTech, Braunschweig, Germany), mouse-anti-human vimentin (1:200, Dako), mouse-anti-human PDGFRα (1:1, Dako), mouse-anti-human CD34 (1:200, Dako). Secondary antibodies were: goat-anti-mouse Alexa 488/594, goat-anti-rabbit Alexa 594, donkey-anti-goat Alexa 488 (MobiTech, Göttingen, Germany). Sections were counter-stained with Dapi and mounted under cover slips with Fluoromount-G (Southern Biotechnology, US). Photos were taken with AxioImagerZ1 (Zeiss, Göttingen, Germany).

6 µm cryosections were prepared and dried overnight prior to fixation in acetone/ethanol. Sections were blocked for 30 min in 4% milk and 15 min in 10% goat serum and incubated with primary Ab diluted in Tris-buffered saline (TBS) for 1 hour at room temperature. Primary Abs were mouse anti-human Kim-1 (Clone 219211; R&D Systems, Minneapolis, MN, USA), mouse anti-human Collagen III (Clone FH-7A; Abcam, Hong Kong) and mouse anti-human Vimentin (Clone V9; Dako, Glostrup, Denmark). Following three 5 min washes in TBS, sections were treated with 1% H₂O₂ in TBS for 10 min to block endogenous peroxidase prior to 30 min incubation in goat anti-mouse horseradish peroxidase. Sections were then washed 3 times in TBS and developed with DAB for 5 min prior to light counterstaining with haematoxylin and eosin.