The immunofluorescence method used in this study was described by Pandurangan et al. [23 (link)]. Paraffin-embedded colonic tissue sections with a thickness of 5 μm were deparaffinized in xylene and then rehydrated in a graded series of ethanol solutions. The slides were then blocked with 5% BSA in TBS for 90 min. The sections were then immunostained with rabbit anti-NF-κB (Santa Cruz Biotechnology, CA, USA) and anti-pSTAT3Y705 antibody (Cell Signaling Technology, CA, USA) diluted 1 : 100 with 5% BSA in TBS and incubated overnight at 4°C. After the sections were washed three times with TBS, the slides were incubated with goat and rabbit DyLight 550 secondary antibody (Thermo Scientific, Rockford, IL, USA) diluted 1 : 200 with TBS and incubated in the dark for 120 min at room temperature. The sections were then washed with TBS and incubated with the nucleus-specific counterstain propidium iodide (Nacalai Tesque, Japan) or 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) to stain the cell nuclei. The slides were mounted in an Ultracruz hard-set mounting medium (Santa Cruz Biotechnology Inc., Dallas, TX, USA), coverslipped, and visualized under a FSX100 fluorescent microscope (Olympus, Tokyo, Japan).
Ultracruz hard set mounting medium
Manufactured by Santa Cruz Biotechnology
Sourced in United States
UltraCruz Hard-set Mounting Medium is a product for use in microscopy. It is designed to provide a durable, long-lasting mounting solution for specimens on microscope slides.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Nacalai Tesque (2 mentions)
Dylight 550 secondary antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Cy3 labeled donkey anti rat igg, by Jackson ImmunoResearch (2 mentions)
A1r confocal microscope, by Nikon (2 mentions)
Anti p stat3 y705 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti nf κb, by Santa Cruz Biotechnology (1 mentions)
Fsx100 fluorescent microscope, by Olympus (1 mentions)
Alexa fluor 488 labeled goat anti human igg, by Thermo Fisher Scientific (1 mentions)
Mec13, by Novus Biologicals (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
P cofilin, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 labeled goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Fv1200 laser scanning confocal microscope, by Olympus (1 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Sc 359850, by Santa Cruz Biotechnology (1 mentions)
Alexa 350, by Thermo Fisher Scientific (1 mentions)
7 protocols using ultracruz hard set mounting medium
1
Immunofluorescence Staining for NF-κB and pSTAT3 in Colon Tissue
2
Immunofluorescence Analysis of Tumor Microenvironment
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To detect TF expression and vasculature, tumor tissues were fixed in 10% paraformaldehyde and sliced tumor sections (10 µm) were stained with 20 µg mL−1 of ALT‐836 and 10 µg mL−1 of CD31/PECAM‐1 antibody (MEC13.3; Novus), respectively. The washed sections were then stained with 5 µg mL−1 of Alexa Fluor 488‐labeled goat anti‐human IgG (Invitrogen) and 5 µg mL−1 of Cy3‐labeled donkey anti‐rat IgG (Jackson ImmunoResearch Laboratories, Inc.). After washing, the sections were mounted with the UltraCruz Hard‐set Mounting Medium containing 1.5 µg mL−1 of DAPI (Santa Cruz Biotechnology). All the fluorescent images were acquired using a Nikon A1R confocal microscope.[54 ] To detect the involvement of the adjacent structures, orthotopic tumors together with thyroid, trachea, muscle, and larynx were carefully resected at necropsy and used for H&E staining as previously described.[51 ]
3
Investigating ROCK1 and LIMK2 Signaling
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The following antibodies served as primary antibodies: rabbit monoclonal antibodies against ROCK1 (Cell Signaling Technology, Danvers, MA, USA, #4035, 1:1000) and phosphorylated-ROCK (p-ROCK; Abcam, Cambridge, UK, #ab203273); rabbit polyclonal antibodies against LIMK2 (Cell Signaling Technology, Danvers, MA, USA, #3845) and phosphorylated-LIMK2 (p-Limk2; Sigma, St Louis, MO, USA, #SAB4300104); mouse monoclonal antibodies against Rho A (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-418, 1:500). Antibodies against Cofilin (#5175) and p-Cofilin (#3311) were purchased from Cell Signaling Technology (Danvers, MA, USA).
EGF was obtained from Worthington Biochemical Corporation (Lakewood, NJ, USA). Regarding specific inhibitors, ROCK1 (Y-27632) was obtained from Selleck (Houston, TX, USA), and Rho Inhibitor I (CT04-A) was purchased from Cytoskeleton (Danvers, MA, USA). Whole Cell Lysis Protein Extraction Kit was purchased from Nanjing keyGEN BioTECH, Ltd. (Nanjing, China). Actin-Tracker Green was obtained from Shanghai Beyotime, Ltd. (Shanghai, China). UltraCruz® Hard-set Mounting Medium was purchased from Santa Cruz Biotechnology (TX, USA, #sc-359850).
EGF was obtained from Worthington Biochemical Corporation (Lakewood, NJ, USA). Regarding specific inhibitors, ROCK1 (Y-27632) was obtained from Selleck (Houston, TX, USA), and Rho Inhibitor I (CT04-A) was purchased from Cytoskeleton (Danvers, MA, USA). Whole Cell Lysis Protein Extraction Kit was purchased from Nanjing keyGEN BioTECH, Ltd. (Nanjing, China). Actin-Tracker Green was obtained from Shanghai Beyotime, Ltd. (Shanghai, China). UltraCruz® Hard-set Mounting Medium was purchased from Santa Cruz Biotechnology (TX, USA, #sc-359850).
4
Visualizing Tumor Microenvironment in Irradiated Mice
5
Immunohistochemical Staining of Colonic CD68
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Paraffin-embedded colonic tissue sections with a thickness of 5 μm were deparaffinized in xylene and then rehydrated in a graded series of ethanol solutions. The slides were then blocked with 5% BSA in TBS for 90 min. The sections were then immunostained with rabbit anti-CD68 antibody (NOVUS Biologicals, Littleton, CO, USA) diluted 1 : 100 with 5% BSA in TBS and incubated overnight at 4°C. After the sections were washed three times with TBS, the slides were then incubated with goat and rabbit DyLight 550 secondary antibody (Thermo Scientific, Rockford, IL, USA) diluted 1 : 200 with TBS and incubated in the dark for 120 min at room temperature. The sections were then washed with TBS and incubated with the nucleus-specific counterstain propidium iodide (Nacalai Tesque, Japan) or 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) to stain the cell nuclei. The slides were mounted in an Ultracruz hard-set mounting medium (Santa Cruz Biotechnology Inc., Dallas, TX, USA), coverslipped, and visualized under a FV1200 laser-scanning confocal microscope (Olympus, Tokyo, Japan).
6
Immunofluorescence Imaging of Transfected Cells
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Transfected cells were grown on 0.1% gelatin-coated glass chip in 24-well plates. Cells were fixed in 4% paraformaldehyde 24 h after initial plating, permeabilized with 2% NP-40, then blocked and incubated overnight with 10% FBS in PBS containing primary antibodies. Primary antibodies were visualized with Alexa-350 (anti-mouse from Life Technologies), Alexa-488 (anti-rabbit, anti-mouse, and anti-goat from Invitrogen) and Alexa-594 (anti-rabbit, anti-mouse, and anti-rat from Invitrogen) secondary antibodies. Nuclei were visualized with 2 μg/ml Hoechst. Slides were mounted with Dako Fluorescent Mounting Medium (Dako) or UltraCruz Hard-set Mounting Medium containing 1.5 μg/ml DAPI (sc-359850, Santa Cruz Biotechnology) and visualized by fluorescence microscopy (Axioplan2, Zeiss; Olympus IX71, Olympus).
7
Alizarin Red S and Annexin V Staining for Mineralization and Phosphatidylserine Detection
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Alizarin red S staining was used to determine the extent of cell matrix mineralization. HDPC was exposed to PSL for 25 days in DIM. PSL treatment was divided into two groups: 1) exposure to PSL for only the first 6 days of cell culture and 2) exposure to PSL throughout the incubation period. Fresh PSL was supplied by exchanging the PSL-containing culture medium at 3-day intervals. To stain calcified nodules, cell cultures were fixed with 3.7% formaldehyde for 24 h, washed with PBS, and then stained with 20 mg/mL alizarin red S for 10 min. The amount of alizarin red S that bound to the cell matrix was quantified by extraction with 10% cetylpyridinium chloride, as described previously 13) .
Annexin V staining of PS PS was localized in PSL-treated HDPC by staining with annexin V-FITC and 4',6-diamidino-2-phenylindole (DAPI). Before staining, HDPC was grown in DIM on culture slides (BD Biosciences, San Jose, CA, USA) for 1, 3 and 6 days in the presence of PSL. The liposomes were replenished at day 3 for 6 days. The cell cultures were then washed with PBS and fixed with 3.7% paraformaldehyde for 20 min. The PS was stained with a FITC annexin V detection kit (BD Biosciences). Cell nuclei were stained with DAPI by UltraCruz Hard-set Mounting Medium (Santa Cruz, CA, USA). Fluorescent images were obtained under a laser scanning confocal microscope (Fluoview FV500, Olympus, Tokyo, Japan).
Annexin V staining of PS PS was localized in PSL-treated HDPC by staining with annexin V-FITC and 4',6-diamidino-2-phenylindole (DAPI). Before staining, HDPC was grown in DIM on culture slides (BD Biosciences, San Jose, CA, USA) for 1, 3 and 6 days in the presence of PSL. The liposomes were replenished at day 3 for 6 days. The cell cultures were then washed with PBS and fixed with 3.7% paraformaldehyde for 20 min. The PS was stained with a FITC annexin V detection kit (BD Biosciences). Cell nuclei were stained with DAPI by UltraCruz Hard-set Mounting Medium (Santa Cruz, CA, USA). Fluorescent images were obtained under a laser scanning confocal microscope (Fluoview FV500, Olympus, Tokyo, Japan).
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