Dfc 480 microscope

Colorectal carcinoma (CRC) tissues were fixed with formalin after surgery and were processed for paraffin embedding. Paraffin-embedded tissues were sectioned and mounted onto glass slides. The slides were deparaffinized and rehydrated. CAIX and PIMT were detected using the EnVision^™ FLEX System (DAKO Agilent, Santa Clara, CA, USA) according to the instructions. Sections were incubated with anti-CAIX antibody M75 (diluted 1:100) or anti-PIMT antibody (NB100-417, Novus Biological, Centennial, Colorado; diluted 1:300) one hour at room temperature. Negative controls were prepared by omission of the primary antibody. The stained sections were examined and photographed with Leica DFC 480 microscope. The study protocol was approved by the Ethics Committee of Biomedical Research Center, Slovak Academy of Sciences, Ethics approval EK/BmV-02/2016 and was in accordance with the principles of the Declaration of Helsinki. All subjects gave their written informed consent before participation.

For whole-mount heart imaging, the hearts from EC-SCL-Cre-ERT^+/−;R26^mTmG/+ mice at E9.5 (n = 3), E10.5 (n = 3) and E12.5 (n = 4) were micro-dissected and fixed in 4% paraformaldehyde for 5 minutes and placed in cold PBS. Images were captured using a Q Imaging RETIGA EXi camera connected to a Leica DMIRE2 microscope.
Immunofluorescence was performed on 5 μm sections of Bouins fixed and paraffin embedded tissues following standard protocols33 (link)34 (link). Primary antibodies used include anti-GFP (1:500 dilution, Abcam, ab13970), anti-PECAM-1 (1:500 dilution, Santa Cruz, sc1506-R), anti-phh3 (1:200 dilution, Cell Signaling, #9701S), anti-cTnT (1:200 dilution, Abcam, ab8295), anti-Hb (1:500 dilution, DAKO, A0118) and anti-Runx1 (1:500 dilution, Abcam, ab23980). The secondary antibodies used were Alexa Fluor 488 goat anti-chicken IgG (H + L) (GFP), Alexa Fluor 568 donkey anti-rabbit IgG (H + L) (PECAM-1, phh3, Hb and Runx1) and Alexa Fluor 568 donkey anti-mouse IgG (H + L) (1:500 dilution; Life Technologies, Carlsbad, CA). Nuclei were stained with Hoechst 33342 (1:1000dilution, Invitrogen, H3570). Images were obtained using a Leica DFC360 FX digital camera connected to a Leica DFC 480 microscope.