Cell inserts were rinsed in PBS, fixed for 10 min in cold 4% PFA in PBS followed by fixation in 1% glutaraldehyde for 30 min at RT. The inserts were cut off with a razor blade and stored in PBS at 4 °C pending sectioning and immunohistochemical analyses. To analyze the bottom side of the inserts, they were placed face down on slides as flat mounts. For staining, slides containing flat mounts or vibratome sections were blocked with 1% BSA and incubated with primary antibodies anti C3/C3b/iC3b/C3d/C3dg (AB11862, Abcam), EFEMP1 (SDIX), MAC (C5b-9 AB55811, Abcam), TIMP-3 (AB39184 Abcam), ELN (sc17581, Santa Cruz Biotechnology), and CFH (AB53800, Abcam) overnight at 4 °C. Secondary antibodies labeled with Alexa-488 or Alexa-555 were incubated for 1 h at RT. Slides were mounted with Fluoromount G and visualized by TCS SP5 II confocal laser scanning microscope (Leica). Samples incubated with 1% BSA instead of primary antibody were used as negative controls.
Fluoromount g
Manufactured by Leica
Sourced in United States
Fluoromount-G is a water-based, permanent mounting medium designed for use in fluorescence microscopy. It is formulated to preserve fluorescent signals and maintain the structural integrity of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Sp8 confocal microscope, by Leica (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab39184, by Abcam (1 mentions)
Ab11862, by Abcam (1 mentions)
Tcs sp5 2 confocal laser scanning microscope, by Leica (1 mentions)
Sc 17581, by Santa Cruz Biotechnology (1 mentions)
Confocal microscope, by Leica (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Nsc23766, by Bio-Techne (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Tcs sp5, by Leica (1 mentions)
Tcs sp2, by Leica (1 mentions)
Type 1 collagen, by BD (1 mentions)
Tcs sp5 multiphoton laser scanning confocal microscope, by Leica (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
8 protocols using fluoromount g
1
Immunohistochemical Analysis of Ocular Tissues
2
Imaging Macrophage Autophagy and Stress
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THP-1-derived macrophages were obtained by seeding 5 × 104 THP-1 cells on glass coverslips in 24-well plates followed by differentiation with PMA for 48 h. After rMS treatment and/or S. aureus infection, macrophages were fixed in 4% PFA in PBS, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 1% BSA in PBS for 1 h, and then incubated overnight at 4 °C with the primary antibodies targeting p62 or phospho-p38 diluted in 1% BSA in PBS. After being washed with PBS, cells were incubated with secondary antibodies diluted in 1% BSA for 1 h at room temperature. Nuclei were counterstained with Hoechst 33342. Coverslips were mounted on slides using Fluoromount-G. Confocal images of p62/SQSTM1 punctate structures were acquired using a Leica SP8 confocal microscope, 40× magnification. Quantitative analyses of fluorescent signals were performed on 10 fields per treatment using Image J v1.53k (National Institutes of Health, Bethesda, MD, USA).
3
Imaging Activated T Cells with DAPI
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TMBP-GFP cells (5 x 105 cells/well) were seeded in 12-well plates together with irradiated rat thymocytes (5000 rad, 1.25 x 107 cells/well) in complete DMEM/1% rat serum with 10 µg/mL MBP and 200 µg/µL PCB for 3 h. Immediately afterwards, the nuclear DNA of T cells was labeled with DAPI following a previously described procedure with minor changes (26 (link)). Briefly, T cells were collected in 15 mL tubes, centrifuged at 400 g, at room temperature for 3 min, washed with PBS 1X (0.5 mL) and fixed during 10 min at 4°C with 4% paraformaldehyde (0.5 mL). T cells were treated first with 0.2% Triton X-100 (0.5 mL) and later with 1 µg/mL DAPI (0.5 mL) at room temperature for 5 and 10 min, respectively, with three washes of PBS 1X (0.5 mL) between them and again after completing the DAPI staining. By using a cytospin device (800 rpm, 3 min), T cells were transferred to microscope slides and cover slips were mounted with Fluoromount-G. Images acquisition was performed with a confocal microscope (Leica) for DAPI (λex=359 nm, λem=461 nm) and PCB (λex=550 nm, λem=591-641 nm) fluorescence. The Fiji software was used for analysis (27 (link)).
4
Confocal Microscopy Imaging of Immunofluorescence
5
Quantification of Neutrophil Extracellular Traps
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Mouse neutrophils were allowed to rest for 1 h, pretreated with vehicle, 20 mM NAC (Sigma-Aldrich, A9165) or 50 mM NSC23766 (To CRIS, 2161) for 0.5 h, and then stimulated with 100 ng/ml PMA (Sigma-Aldrich, P1585), 20 μM hemin (Frontier, H651-9) and MOI = 1 iRBCs purified as described above for 4–6 h. An equal volume of PBS or DMSO was added as a control. Then, the cells were stained with 0.5 μM Sytox Green for 10 min. Following remove of the supernatant and washing twice, the cells were fixed with 4% PFA in PBS for 15 min at RT. After another wash, the cells were stained with DAPI Fluoromount-G™ and images were obtained with a Leica SP8 confocal microscope using a 63× oil objective lens. For quantification, NETs were counted in at least five random fields per sample.
6
Chromatin Structure Analysis by MNase Assay
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Fresh isolated nuclei were obtained as above. Samples were resuspended in 300 µl of buffer B and 2.5
Units/50 µl of MNase were added. Reactions were performed at 37°C and stopped at 0, 5, and 10 min by adding 3 l of 0.5 M EDTA. Nuclei were then fixed in 4% PFA for 5 min, spotted on a glass coverslip and stained with DAPI for 10 min at RT. Nuclei were mounted with Fluoromount-G and imaged using a Leica SP5 confocal microscope. DAPI intensity and heterochromatin area were calculated by using ImageJ software.
Units/50 µl of MNase were added. Reactions were performed at 37°C and stopped at 0, 5, and 10 min by adding 3 l of 0.5 M EDTA. Nuclei were then fixed in 4% PFA for 5 min, spotted on a glass coverslip and stained with DAPI for 10 min at RT. Nuclei were mounted with Fluoromount-G and imaged using a Leica SP5 confocal microscope. DAPI intensity and heterochromatin area were calculated by using ImageJ software.
7
Retinal Immunohistochemistry and Imaging
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Fixed whole-mount retinas were permeated with 2% (wt/vol) Triton X-100 in PBS, blocked in BGT (2.5% (wt/vol) BSA, 100 mM glycine, 0.25% (wt/vol) Triton X-100 in PBS) and incubated overnight at 4 °C, with the indicated primary antibodies or 2 h at RT with a biotinylated probe, after washing with TBS. Retinas were then washed with PBS and incubated for 2 h at RT with the secondary antibodies or streptavidin. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, MA USA), the retinas were mounted in Fluoromount-G and analyzed with a laser confocal microscope (TCS SP5 and TCS SP2; Leica Microsystems). Antibodies are listed below.
For determining microglia density, serial optical sections were acquired with a × 40 objective every 2.5 μm through the whole retina in four central fields around the optic nerve. To measure the percentage of the area occupied by GFAP staining, serial optical sections were acquired with a × 40 objective every 2 μm, covering the whole ganglion cell layer in 12 fields (four central, four medial and four peripheral) around the optic nerve. Confocal maxima of the ganglion cell layer Z sections were then converted into a black and white image and analyzed with the FIJI Software to calculate the percentage of total area filled with GFAP.
For determining microglia density, serial optical sections were acquired with a × 40 objective every 2.5 μm through the whole retina in four central fields around the optic nerve. To measure the percentage of the area occupied by GFAP staining, serial optical sections were acquired with a × 40 objective every 2 μm, covering the whole ganglion cell layer in 12 fields (four central, four medial and four peripheral) around the optic nerve. Confocal maxima of the ganglion cell layer Z sections were then converted into a black and white image and analyzed with the FIJI Software to calculate the percentage of total area filled with GFAP.
8
Immunofluorescence Imaging of Cells in 2D and 3D
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