Alexa fluor goat anti rabbit igg

Western blotting was performed as described previously ^{8 (link), 61 (link)}, with the exception that Alexa Fluor secondary antibodies were used. All blots were developed using the Odyssey fluorescence image scanner and the band intensities were quantified using LI–COR software. Cell cycle analysis was performed using PI (propidium iodide) staining as described previously ^{8 (link)}, using the CycleTEST PLUS DNA reagent kit (BD Biosciences, San Jose, CA, USA; # 340242). The following antibodies were used in the study: anti–P53 (SCBT; sc–6243), anti–P21 (BD; #556431), anti–APAF1 (R & D; #MAB828), anti–PIG3 (abcam; #ab64798), anti–PUMA (CST; #4976); anti–Ash2L (Bethyl Labs; #A300-108A), anti–H3K4me3 (abcam; #ab1012), anti–Wdr5 (abcam; #ab56919); anti–RbBP5 (Bethyl Labs; #A300-109A), anti–RNAP II (SCBT; sc–899); anti–Serine5–CTD (abcam; #ab5131–50); anti–TAFII (SCBT; sc–735); anti–TFIIB (SCBT; sc–225); anti–TFIIF (SCBT; sc–235); anti–Tubulin (Sigma; #T5168), Alexa Fluor 680 goat anti–mouse IgG (Molecular Probes; #A21057) and Alexa Fluor goat anti–rabbit IgG (Molecular Probes; #A21076).

The mouse stomach was cut into 2–3 mm pieces and then incubated in 5 mM EDTA for 30 minutes, with shaking at 37°C. The cell suspension passed through 40 μm cell strainers 2 times. After centrifuging at 1200 RPM for 5 minutes, the pellet was suspended in 50 μL Matrigel (BD Biosciences) and plated in pre-warmed 24-well plates. The 24-well plate was placed in a CO₂ incubator for 20 minutes to allow a complete polymerization of the Matrigel, 500 μL mouse basal medium (STEMCELL, Cambridge, MA) was overlaid. Organoids were collected after 14 days, fixed in 4% paraformaldehyde for 20 min, washed in 1× PBS, and re-suspended in 30 μL Histo-gel (Richard-Allan, White Plains, NY). The histo-gel was placed into a cassette and fixed in 70% ethanol. Paraffin-embedded organoids were pretreated at 65°C for 90 min, followed by deparaffinization using standard procedures.^{60 (link)} Antigen retrieval was performed using Tris EDTA Buffer (Genemed, Torrance, CA). Slides were then incubated for primary antibody overnight at 4°C, and further incubated with both Alexa Fluor goat anti-rabbit IgG and Alexa Fluor goat anti-mouse IgG (1:800) (Invitrogen) at room temperature for 2 h. Images were acquired using an inverted laser-scanning LSM880-Airyscan (Zeiss, Thornwood, NY) confocal microscope.

To demonstrate the proximity (<40 nm) between two different proteins in gastric cancer cells, PLA was performed by using Duo-link In Situ-Fluorescence kits according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO). The slides were mounted with the mounting solution with DAPI. Each dot represents the proximity of two interacting proteins. Cell images were acquired using an Inverted laser-scanning LSM880-Airyscan (Zeiss, Thornwood, NY) confocal microscope.
Following cell fixation and permeabilization, immunofluorescence was performed using standard methods.^{60 (link)} The cells were washed with cold PBS three times and incubated with both Alexa Fluor goat anti-rabbit IgG and Alexa Fluor goat anti-mouse IgG (1:800) (Invitrogen) at room temperature for 1.5 h. Cell images were acquired using an Inverted laser-scanning LSM880-Airyscan (Zeiss, Thornwood, NY) confocal microscope.