Sections were fixed in 4% paraformaldehyde or methanol carnoy (60%methanol, 30% chloroform, 10% acetic acid), paraffin embedded, sectioned, and stained with hematoxylin and eosin or periodic acid-schiff/Alcian blue. For immunofluorescence, tissue was stained with anti-GFP (ThermoFisher), anti-IL-18 (Rockland), or HCS CellMask™ Red Stain (ThermoFisher) and DAPI. For histological scoring, H&E-stained colonic tissue sections were blindly graded by a board-certified pathologist on a scale of 1–5 for each of the following C. rodentium-associated histologic parameters: (a) mural edema (1–5), (b) epithelial lesions (crypt hyperplasia, elongation, erosion) (1–5), and (c) leukocyte infiltration (1–5) (Giacomin et al., 2015 (link); Jain et al., 2015 (link); Liu et al., 2012 (link); Osborne et al., 2013 (link)). Each parameter was scored at 12 days post infection on a scale ranging from 1–5; allowing for maximum total histologic score of 15. 1 corresponded to no inflammation and 2, 3, 4, and 5 corresponded to minimal, mild, moderate, and severe, respectively.
Hcs cellmask red stain
Manufactured by Thermo Fisher Scientific
The HCS CellMask™ Red Stain is a fluorescent dye that selectively stains the plasma membranes of cells. It can be used to visualize and distinguish individual cells within a population.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342 stock solution, by Thermo Fisher Scientific (1 mentions)
Hydrogen peroxide solution, by Merck Group (1 mentions)
Alexa 647 anti mouse, by Thermo Fisher Scientific (1 mentions)
Operetta high content imaging system, by PerkinElmer (1 mentions)
5 protocols using hcs cellmask red stain
1
Histological Evaluation of Colitis in Mice
2
Immunofluorescent Staining of Porcine Gastric Mucin
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5 µm thick tissue sections were cut from porcine gastric tissue specimens fixed in Carnoy's solution and embedded in paraffin. Sections were deparaffinized and rehydrated in sequential washes of 95%, 80% and 50% ethyl alcohol, and antigen retrieval was performed using 10 mM sodium citrate (pH 6.0) at 95°C for 10 min. Nonspecific binding was blocked with 1% BSA for 1h. Primary antibody anti-MUC5AC (45M1; Sigma-Aldrich,M5293) was diluted 1:1,000 and incubated at 4°C for 3 h. Sections were washed with cold PBS (140 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.4) 3 times and incubated with anti-mouse secondary antibody conjugated with Alexa Fluor 350 (Life Technologies, A11045), diluted 1:200 at 4°C for 3 h. Sections were incubated with HCS CellMask™ Red Stain (ThermoFisher, H32712) diluted 1:14,000 at RT for 30 min. Sections were washed again with cold PBS 2 times and with water one time and mounted with ProLong® Gold Antifade Mountant (Life Technologies, P36934).
3
Antibody Labeling and Cellular Imaging
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All conjugated and unconjugated primary antibodies used in this study are listed in Table 2 . Indirect immunofluorescence was performed using secondary antibodies conjugated with Alexa-647 anti-Mouse (Invitrogen, Cat. A-21236), Alexa-555 anti-Rat (Invitrogen, Cat. A-21434) and Alexa-488 anti-Rabbit (Invitrogen, Cat. A-11034). 10 mg/ml Hoechst 33342 stock solution was purchased from Life Technologies (Cat. H3570). 20xPBS was purchased from Santa Cruz Biotechnology (Cat. SC-362299). 30% hydrogen peroxide solution was purchased from Sigma-Aldrich (Cat. 216763). PBS-based Odyssey blocking buffer was purchased from LI-COR (Cat. 927–40150). All reagents for the Leica BOND RX were purchased from Leica Microsystems. HCS CellMask Red Stain and Mito-tracker Green stains were purchased from ThermoFischer (catalog numbers H32712, R37112 and M751, respectively).
4
Senescence-Associated β-Galactosidase Staining
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SA-β-Gal staining was performed using the Cellular Senescence Detection Kit—SPiDER-ßGal (Dojindo) as per the manufacturer’s instructions. Briefly, Bafilomycin A1 working solution was added to the plate and incubated at 37 °C and 5% CO2 for 1 h. The solution was discarded and replaced with SPiDER-βGal working solution and incubated at 37 °C and 5% CO2 for 30 min. Cells were washed twice before staining with Hoechst 33,342 (Invitrogen) and HCS CellMask™ Red Stain (Life Technologies). Plates were incubated at 37 °C and 5% CO2 for 1 h and observed under a fluorescent microscope.
5
Virus Infection and Imaging of CHO Cells
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CHO-derived cell lines Par6, FD11, and FD11-Fur, seeded in MEM-alpha medium with 5% FBS, were infected with recombinant CCHFV WT or RVFV-ΔNSs:GFP-ΔNSm [34 (link)], and fixed in 10% formalin buffered solution. CCHFV-infected cultures were incubated with CCHFV HMAF and goat anti-mouse Alexa 488-conjugated antibody. Nuclei and cells were respectively counterstained with Hoechst 33342 and HCS CellMask Red stain (Life Technologies). Images were acquired with a 20× objective on an Operetta High Content Imaging system (PerkinElmer Inc, Waltham, MA, USA). To detect RVFV-infected cells, EGFP was measured.
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