For immunofluorescence HeLa cells were plated onto coverslips prior to treatments. Following treatments, cells were washed three times in PBS and fixed using 4% PFA in DPBD for 10 mins. Cells were then permeabilised for 10 mins in 0.2% Trition X-100 PBS. Coverslips were then incubated in blocking buffer (1% BSA TBS) for 1 hour. For immunostaining, coverslips were inverted on to droplets of blocking buffer containing Phospho-Histone H2A.X (Ser139) (CST, 2577) antibody (1:500) then incubated in a humidified chamber overnight at 4°C. Subsequently, coverslips were washed 3 times in PBS + 0.1% Tween then incubated at room temperature in the dark for 1 hour in blocking buffer containing Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen, A-11035) secondary antibody (1:1500) for 1 hour. Coverslips were washed three times TBS + 0.1% Tween, nuclei were stained with 300 nM (100 ng.mL-1) Hoechst 33342 for 15 mins. Coverslips were then washed three times in TBS, rinsed briefly in distilled water and mounted using Fluoromount-G Mounting Medium (INTERCHIM). All images were acquired using a Zeiss Axio Observer spinning-disk confocal microscope driven by the MetaMorph software. For quantification a minimum of ten fields of view were obtained per condition of each biological replicate.
Fluoromount g mounting medium
Manufactured by Interchim
Fluoromount-G is a water-based mounting medium designed for use with fluorescent-labeled samples. It is formulated to preserve fluorescent signals and provide long-term stability for microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer, by Zeiss (3 mentions)
Alexa fluor 546 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Bitplane, by Oxford Instruments (1 mentions)
Axio observer z1 inverted microscope, by Zeiss (1 mentions)
Superfrost plus slides, by Leica (1 mentions)
Application suite x software, by Leica (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Imagej, by Zeiss (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (1 mentions)
5 protocols using fluoromount g mounting medium
1
Immunofluorescence Labeling of Phospho-Histone H2A.X in HeLa Cells
2
Quantifying Bacterial Membrane Depolarization
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Cocultures were prepared as indicated previously. The dye DiBAC4(3) was added to the BHI medium (final concentration 2.5 μM) when the cocultures were prepared. Nisin was used as a positive control (final concentration 5 μM). After 3, 4, and 6 h of coculture, bacteria were washed once and resuspended in PBS, mounted on a glass coverslip with Fluoromount-G mounting medium (Interchim), and dried in the dark at 37 °C for 30 min. Slides were observed with an AxioObserver.Z1 inverted microscope (Carl Zeiss) equipped with a high-speed CSU-X1 spinning-disk confocal system (Yokogawa) and an Evolve EM-CCD camera (Photometrics). Images were acquired through a Plan-Apochromat 100× oil objective, using MetaMorph software (version 7.7.9.0). Stacks of 20 images were acquired every 200 nm in the z axis. Three-dimensional reconstruction was performed on stacks with IMARIS software (Bitplane; Oxford Instruments). Icy was used for image processing and analysis. For each condition tested, at least 150 cells were marked as regions of interest (ROIs) manually by using only one image of the Z stacks (better focused image). To quantify the effects of depolarization, ROI mean pixel intensities of the GFP channel were obtained and normalized to the background mean pixel intensities of the GFP channel.
3
Immunohistochemical Analysis of Rogdi
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Embryos (E12.5 to E18.5) and postnatal day (PN) 1, 3, 5, 7 and 14 heads were freshly fixed in 4% paraformaldehyde for 24 h, demineralized with 10% EDTA (postnatal stages), cryoprotected with 20% sucrose, and embedded in Shandon Cryomatrix Frozen Embedding Medium (Thermo Scientific™). Frozen sagittal Sects. (10 μm) were cut using a Leica CM3050 S cryostat and placed on Superfrost Plus™ slides for immunohistochemistry. Antigen retrieval was performed according to the manufacturer’s antibody protocol. The samples were blocked with 5% normal donkey serum (NDS) or normal goat serum (NGS) in 0,05% TBS Tween 20 (TBSTw). Sections were incubated with anti-Rogdi (Proteintech® 17047-1-AP) in blocking solution (1:100). After washing with TBSTw, samples were incubated with secondary Ab labeled with fluorophore in TBS (1:500) and DAPI (5 mg/ml) to a final dilution of 1:5000–10,000. Sections were mounted with FluoroMount-G Mounting medium (FP-483331, Interchim). Images were acquired with an upright motorized microscope (Leica DM 4000 B) equipped with a Photometrics CoolSNAP HQ2 camera and analyzed in Leica Application Suite X software.
4
Microscopic Observation of L. monocytogenes
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Observation of intracellular L. monocytogenes by microscopy was performed as in (Bierne et al., 2021 (link)). Briefly, cells grown on 12 mm or 22 mm coverslips were rinsed in 1× phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) in PBS for 30 minutes at room temperature. Cells were washed in PBS, permeabilized in 0.4% Triton X-100 in PBS, washed 3 times in PBS and incubated in blocking solution (2% bovine serum albumin (BSA) in PBS), before being processed for immunofluorescence, successively with the primary antibody solution, and secondary antibody solution containing Alexa fluor 647-conjugated phalloidin and Hoechst (in 2% BSA). Coverslips were mounted on glass slides using Fluoromount-G mounting medium (Interchim, Montlucon). Samples were analyzed with a Carl Zeiss AxioObserver.Z1 microscope equipped with 20× non-oil immersion or 40×, 63× and 100× oil immersion objectives connected to a CCD camera. Images were processed using ZEN (Carl Zeiss) or ImageJ.
5
Immunofluorescence Analysis of SIRT2-GFP Translocation
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For immunofluorescence HeLa cells were plated onto coverslips prior to treatments. Following treatments cells were washed three times in PBS and fixed using 4% PFA for 10 mins. Cells were then permeabilised for 10 mins in 0.1% Trition X-100 PBS. Nuclei were stained with 300 nM (100 ng.mL−1) DAPI for 15 mins, then washed three times in PBS before being mounted using Fluoromount-G® Mounting Medium (INTERCHIM). For live imaging HeLa cells were plated onto 35 mm glass bottom dishes (MatTek, P35G-1.5-10-C) in FluoroBrite™ DMEM (Gibco) supplemented with 10% FCS. Cells were treated with 20 nM LMB and images were acquired every 5 mins in an atmospheric chamber at 37 °C. All images were acquired using a Zeiss Axio Observer spinning-disk confocal microscope equipped driven by the MetaMorph software. For quantification a minimum of ten fields of view were obtained per condition of each biological replicate and the nuclear to whole cell ratio of SIRT2-GFP was determined for individual cells. Image analysis of nuclear translocation following siRNA transfections was carried out using CellProfiler48 (link) and is expressed as the mean nuclear fluorescence intensity of SIRT2-GFP over the mean fluorescence of the whole cell.
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