Confocal tcs sl dmre microscope

Tumor tissues were embedded in OCT medium-containing cryomolds and immediately frozen in 2-methyl-butane. Then, 5 μm frozen tissue sections were cut using a cryostat and layered on super frost plus slides. Cells were grown on glass coverslips in 12 well plates for in vitro experiments. The covers were incubated in 4% paraformaldehyde for 15 min and then washed and blocked for 60 min at room temperature. The cells were then incubated with MUC2 or GRP78 or CHOP antibody. They were then washed 5 times with 1X PBS and incubated with anti-rabbit Alexa 647 and anti-mouse Alexa 488 and SYTOX orange for nucleic acid staining at room temperature for 30 min. Repeat 5 times washing with 1X PBS was performed. Glass slips were mounted on slides using ProLiong Gold antifade solution from Invitrogen (Life Technologies, Grand Island, NY). Confocal images were taken from 10 different fields at random at X63 magnification using a LEICA confocal TCS SL DMRE microscope.

Cells or tissue were placed in Tissue Path Disposable Base Molds and snap frozen in Tissue-Tek O.C.T compound. Using a cryostat microtome, 5 micron frozen sections of tumor tissue were mounted on Superfrost Plus microscope slides and maintained at –20°C. The slides were incubated in 4% paraformaldehyde for 15 minutes, washed, and blocked for 60 minutes at room temperature. The slides were then stained for 3 hours at room temperature with MUC2 or COX-2 antibody. The slides were washed 3 times with 1X PBS and incubated with anti-rabbit Alexa 647 or Alexa 488 and SYTOX Orange for nucleic acid staining for 30 minutes at room temperature. The slides were washed 3 times with 1X PBS and once with high-salt PBS. Cover slips were mounted on the sections using ProLong Gold antifade solution from Invitrogen (Life Technologies, Grand Island, NY). In situ apoptosis in explant tissue, colonoid cultures and LS174T cells was detected by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) using in situ BrdU-Red DNA fragmentation assay kit (ab66110, Abcam, Cambridge, MA) according to the manufacturer’s protocol. Confocal images were randomly taken of 10 different fields (X 63 magnification) using a LEICA confocal TCS SL DMRE microscope. Images of each slide were then analyzed using Image-pro Premier Software to quantify the average intensity of MUC2 or COX-2 expression.