Efficient navigation zen software

Tumor tissue (n = 4–5 mice/group) was fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 24 h, then transferred and stored in 70% ethanol. Paraffin-embedded sections were deparaffinized with xylene and rehydrated through a series of descending concentrations of ethanol. Antigen retrieval was performed by using recombinant RT-PCR grade Proteinase K (20 µg/ml; Roche). Endogenous peroxidase and proteins were blocked with 3% H₂O₂ and Tris-NaCl–blocking buffer, respectively. Slides were incubated with purified rat anti-mouse CD31 (BD Pharmingen, San Jose, CA, USA) overnight at 4°C. Mouse absorbed biotinylated anti-rat secondary Ab (Vector Laboratories, Burlingame, CA, USA) was used. TSA Biotin System (PerkinElmer, Waltham, MA, USA) was used for signal amplification, and the ImmPact DAB Peroxidase Substrate Kit (Vector Laboratories) was used to visualize staining. Tissues were counterstained with hematoxylin and mounted with permount (Thermo Fisher Scientific). A Zeiss A1 Scope and AxioCam ICc5 and Zeiss Efficient Navigation (ZEN) software (Zeiss, Oberkochen, Germany) was used to obtain images of slides after staining (5–10 fields/tumor). ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to quantify the number of vessels per field.

Differential interference contrast (DIC) images and fluorescence images were obtained with a BX71 microscope (Olympus, Tokyo, Japan), a Quantix 1400 camera (Photometrics), and IPLab 3.7 software (Scanalytics). In observation of Osh2-P4M, images were obtained with a BZ-X710 microscope (Keyence, Osaka, Japan), and processed with Fiji (ImageJ; National Institutes of Health, http://rsb.info.nih.gov/ij/). PSM perimeter measurements were performed as previously described [34 (link)]. PSM perimeters were measured by Fiji. Confocal images were obtained with a ZEISS LSM 880 with Airyscan (Carl Zeiss, Oberkochen, Germany) with ZEISS Efficient Navigation (ZEN) software (Carl Zeiss).

Flat-mounted cornea IHC of corneas was performed by following procedure: (i) fixed corneas were washed with 1X PBS for 24 h at 4 °C with shaking, (ii) transferred to 1% Triton X-100 (Sigma-Aldrich) in 1X PBS (1% PBST) and incubated for 24 h at 4 °C with shaking, (iii) immersed in 5% NGS-based blocking buffer for 24 h at 4 °C with shaking, (iv) immersed in fluorophore-conjugated primary antibody solution at 4 °C for 48 h with shaking, (v) washed with 1% PBST for 1 h at room temperature with shaking twice, (vi) Specimens were placed on a slide glass and mounted with mounting medium (ProLong^TM Gold, Invitrogen). Anti- α-SMA-Alexa 647 (1:100 dilution, sc-53015-AF647, Santa Cruz) and anti-COL3A1-Alexa 488 (collagen type III, 1:100 dilution, sc-271249-AF488, Santa Cruz) antibodies were used for flat-mount IHC. All fluorescence images were taken with Zeiss Axio Scan.Z1 (Carl Zeiss). The area of collagen type III and α-SMA expressed region was quantified with threshold-based area measuring method using Zeiss Efficient Navigation (ZEN) software (desk, version 2.3, Carl Zeiss).