Dmra fluorescent microscope

Immunohistochemical (IHC) analyses were performed as previously described (Liang et al., 2012 (link); Chagani et al., 2017 (link); Li et al., 2017 (link)). Briefly, sections of 5 μm thickness were rehydrated and dissolved of paraffin with a graded xylene and ethanol series. Antigen retrieval was done with a citrate buffer pH 6.0. Blocking was done with 10% normal goat serum in PBS. Primary antibodies used were anti-CPD (Abcam, 1:1000) and anti-PCNA (Abcam, 1:6000) with incubation overnight at 4°C. Cy3 (Jackson Immuno Research, 1:400) was used as the secondary antibody. Nuclear staining was done using DAPI (0.2 ng/mL). Samples were then rinsed with PBST, dehydrated with graded ethanol and xylene washes, and mounted with distyene plasticizer xylene (DPX). Slides were allowed to dry overnight at room temperature before capturing images with Leica DMRA fluorescent microscope and Hamamatsu C4742-95 at 20x magnification using AxioVs40 version 4.8.2.0 software. Images were processed using Adobe Photoshop CC 2018 and Image J software version 1.50i.

To obtain stacked images, specimens were examined and photographed under a Confocal Zeiss Axio Imager microscope (with apotome) using an excitatory wavelength of 488 nm. Number of Z slices and depth of Z slice interval depended on the topology and thickness of tissue. Snap shot images were obtained using a Leica DMRA fluorescent microscope with Hamamatsu GRCA-ER digital camera or Confocal Zeiss Axio Imager microscope. Images were processed using Axiovision 4.8.1 software. Light microscope images were obtained using Leica M205 C.