Dxm1200f camera

RPE cells from the primary cultures were seeded on 22 mm glass coverslips and allowed to reach confluence for 2 weeks with the medium (Opti-MEM containing 4% FBS) changed every 4 days. Cells were fixed in 4% paraformaldehyde for 10 min, washed with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4), and permeabilized with 0.1% Triton X-100 for 5 min, and then blocked for 1 h with either PBS-Tween-0.05%, FBS 10% (for zonula occludens 1 [ZO-1]), PBS-Tween-0.05%, FBS 10%, 1% BSA (for RPE), and PBS-casein 0.5%-saponin 0.1% (for CRALPB). Primary antibody was incubated overnight at 4 °C in blocking buffer. For ZO-1 (Santa Cruz, Santa Cruz, CA, Cat No sc33725), the antibody was diluted 1:50, RPE65 (Novus Biologicals, Centennial, CO, Cat No NB100355) 1:250, and CRALPB (Abcam, Cambridge, UK, Cat No 15051), 1:50. Fluorescein isothiocyanate (FITC)-labeled secondary antibodies were incubated in blocking buffer 1:100 for ZO-1 and cellular retinaldehyde-binding protein (CRALBP); 1:500 for RPE65. The cells were washed three times with PBS for 5 min. Samples were mounted with Dako Fluorescence Mounting Medium (Dako North America, Inc., Carpinteria, CA), and visualized using a microscope Eclipse TE 2000-U. Images were acquired with a DXM1200F camera and 40X objective (0.6 NA) using ACT-1 software (All from Nikon, Tokyo, Japan).

FFPE colon sections were probed with antibodies against F4/80 (AbD Serotec), Gr-1 (AbD Serotec), CD3 (Abcam), B220 (BD), p-STAT3 (Cell Signaling Technology), Vimentin (Abcam), and α-SMA (FITC-conjugated; Sigma-Aldrich). The anti–rabbit Alexa Fluor 568–conjugated secondary antibody (Invitrogen), biotinylated secondary antibodies (Vector Laboratories), the Vectastain ABC kit (Vector Laboratories), and the Vectastain DAB kit or the Vectastain NovaRED kit (Vector Laboratories), were used for signal amplification and detection. Images were acquired with an Eclipse E800 microscope (Nikon) equipped with a Dxm1200F camera (Nikon). Cryosections of paraformaldehyde (PFA)-fixed tissue samples or PFA-fixed cells were probed with antibodies against Vimentin (Abcam), α-SMA (Sigma-Aldrich), and Collagen IV (Abcam), followed by secondary antibodies conjugated with Alexa Fluor 647 (Invitrogen). Images were acquired with a TCS SP8X White Light Laser confocal system (Leica).

Tumors were washed with PBS and preserved in 10% formalin for 24–48 hours and in 70% ethanol until paraffin embedding. Sections of 4.5 µm thickness were deparaffinized in xylene for 6 min, then washed in 50% xylene in ethanol and 100% ethanol, and rehydrated by sequential transfer to 95%, 70%, and 50% ethanol, and ultimately to distilled water for 3 min each before staining. Samples were washed in PBS, blocked in 1% bovine serum albumin for 30 min, and incubated with rabbit polyclonal anti-CD8a antibody to CD8a antibody (synaptic system) at 4°C overnight. Samples were then washed and blocked in goat serum, incubated with biotin-conjugated goat antirabbit IgG for 45 min, washed for 30 min in PBS, then incubated with horseradish peroxidase (HRP)-conjugated avidin. Samples were developed using the CN/DAB Substrate Kit (Thermo Fisher, Waltham, Massachusetts) for 5 min as per vendor instructions, then counterstained with hematoxylin for 10 s, and washed and mounted. Samples were visualized using a 10× or 20× objective on a Nikon Eclipse E400 microscope, and images were captured using a DXM1200F camera using the software provided by the vendor (Nikon, Melville, New York). At least 10 images from each tumor sample were obtained for each magnification. Cells were counted using ImageJ software after appropriate background subtraction.