Fifty thousand Vero E6 cells were seeded on the top of 13 mm circular cover slips in a 24-well plate in M10 media and incubated overnight at 37 °C and 5% CO2. Therefore, cells were infected with CHIKV (MOI 0.1), and incubated for 20 h under the same conditions. The medium was removed, and the cells were fixed with 4% paraformaldehyde solution for 30 min. After 3 washes with PBS (5 min/wash), the cover slips were incubated with pooled mouse sera (1:500) for 1 h. After an additional wash with PBS, the cover slips were incubated with donkey anti-mouse IgG conjugated with Alexa 488 (1:500, Invitrogen, Waltham, USA) for 30 min. After another wash with PBS, cover slips were incubated with DAPI (1:1000, Invitrogen, Waltham, USA). For actin staining, the samples were stained with phalloidin–Texas Red (Invitrogen) according to the manufacturer’s instructions and previous work [90 (link)]. Finally, the stained cells were washed 3 times with PBS and mounted with the anti-quenching Fluoromount-GTM mounting medium (ThermoFisher, Waltham, MA, USA) on glass slides. Images were obtained using a Leica-SP8 confocal microscope. Z images were acquired with a 0.15 μm spacing between the sections, and all images were treated with the open-source free software ImageJ version 1.46r.
Fluoromount gtm mounting medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluoromount-G™ is a water-soluble, synthetic mounting medium designed to preserve fluorescence in immunofluorescence applications. It provides a permanent, non-hardening mount for fluorescently-labeled specimens.
Automatically generated - may contain errors
Lab products found in correlation
Texas red phalloidin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Sytox orange, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Inverted epifluorescence microscope, by Olympus (1 mentions)
Cellsens software, by Olympus (1 mentions)
Rhodamine phalloidin, by Merck Group (1 mentions)
4 protocols using fluoromount gtm mounting medium
1
Immunofluorescence Analysis of CHIKV-Infected Vero Cells
2
Bovine PMN-Spermatozoa Interaction Imaging
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Bovine PMN and spermatozoa (1:1) were incubated for 120 min at 37 °C in 5% CO2, fixed with 4% p-formaldehyde for 15 min, washed with PBS, and blocked in PBS supplemented with 2% BSA (PBS-BSA) for 30 min at RT. The samples were incubated overnight with a 1:300 dilution of rabbit anti-bovine ELA polyclonal IgG antibody (Abcam, Cambridge, UK) in PBS-BSA at RT, washed three times with PBS at 100 rpm of shaking for 5 min, and then incubated for 1 h with a 1:500 dilution of goat IgG anti-rabbit IgG secondary antibody conjugated with Alexa FluorTM 488 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) in PBS-2% BSA at RT. The samples were washed three times with PBS with 100 rpm in a Classic Orbital Shaker (DLAB, Beijing, China) for 5 min, and incubated with a 1:200 dilution of SYTOX© Orange (Invitrogen) for 15 min at RT for DNA staining. Finally, samples were washed with PBS and mounted with Fluoromount-GTM mounting medium (Thermo Fisher Scientific, Waltham, MA, USA) for their visualisation via microscopy using a TissueFAXS i Plus Cytometry (TissueGnostics, Vienna, Austria).
3
Immunofluorescence Analysis of hCECs
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The hCECs were seeded in 384-well plates pre-coated with iMatrix-511 at a ratio of 500 cells/mm2 and cultured in BGM alone (control) or BGM containing TGF-β1, 2, or 3, or one of the TGF-β cell signaling pathway inhibitors, for four weeks. The IF protocol was previously described by our team [29 (link),42 (link)]. Briefly, cells were fixed in pure methanol at room temperature for 15 min after rinsing with PBS containing Ca2+ and Mg2+. The cells were then rehydrated in PBS and incubated in blocking buffer (PBS, 2% bovine serum albumin, 2% goat serum) for 30 min at 37 °C. The primary antibodies, diluted to 1/300 in blocking buffer, were incubated with cells at 37 °C for one hour under gentle agitation (30 rpm). After three rinses in PBS, the secondary antibodies, diluted to 1/600, and DAPI diluted at 2 µg/mL in blocking buffer were incubated with cells at 37 °C for one hour under gentle agitation. After three rinses in PBS, the cells were immersed in Fluoromount-GTM mounting medium (00-4958-02, Invitrogen) to protect the fluorochromes (Alexa Fluor™ 488 and DAPI). An epifluorescence inverted microscope (IX81, Olympus, Tokyo, Japan) with the CellSens software (Soft Imaging System GmbH, Olympus) was used to acquire images.
4
Quantifying Cell Morphological Changes Induced by Toxins
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A monolayer of Vero cells was obtained by seeding 1 mL of a 1 × 105 cells mL−1 suspension per well in a 24-well plate (TPP), followed by incubation for 24 h at 37 °C with 5% CO2. Sub-confluent cells were then incubated for 18 h with 4 µg.mL−1 of native toxins (positive controls) or purified recombinant toxins. In addition, a negative control was also added by keeping cells in DMEM and 0.1% BSA only. After incubation, cells were fixed and stained with Rhodamine Phalloidin (reference: P1951, Sigma Aldrich, St. Louis, MO, USA) and DAPI contained in the Fluoromount-GTM mounting medium (reference: 00495952, Invitrogen, San Diego, CA, USA). Five random fields were acquired for each condition on a LEICA SP8 confocal microscope using ×20 objective, and FIJI software version 2.9.0/1.53t was used to determine the mean area per cell [49 (link)]. For each field randomly selected, using a thresholding method and particle analysis, we used the actin channel to determine the area of the field occupied by cells, expressed in µm2, and the DAPI channel to count the number of nuclei in the field. We then expressed the average cell area, expressed in µm2/cell, by dividing the total cell area by the number of cells. Finally, additional images were acquired with the ×63 objective in order to illustrate cell morphological alterations induced by toxins.
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