Fluorsave reagent

Manufactured by Merck Group

Sourced in United States, Germany, United Kingdom, Switzerland

FluorSave Reagent is a laboratory product manufactured by Merck Group. It is a mounting medium designed for the preservation and long-term storage of fluorescently labeled specimens, such as those used in fluorescence microscopy. The reagent helps to maintain the fluorescence signal and prevent fading of the labeled samples.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (20 mentions) Hoechst 33342, by Thermo Fisher Scientific (14 mentions) Lsm 710 confocal microscope, by Zeiss (12 mentions) Bovine serum albumin (bsa), by Merck Group (9 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions) Triton x 100, by Merck Group (6 mentions) Lsm 700 confocal microscope, by Zeiss (6 mentions) Paraformaldehyde (pfa), by Merck Group (5 mentions) Rhodamine phalloidin, by Thermo Fisher Scientific (5 mentions) Lsm 710, by Zeiss (4 mentions) Hoechst, by Thermo Fisher Scientific (4 mentions) Hoechst 33258, by Merck Group (4 mentions) Lsm 700, by Zeiss (4 mentions) Lsm 880 airyscan inverted confocal microscope, by Zeiss (3 mentions) Microm hm550 cryostat, by Thermo Fisher Scientific (3 mentions) Apoptag fluorescein in situ apoptosis detection kit, by Merck Group (3 mentions) Hoechst, by Merck Group (3 mentions) Hoechst 33342, by Merck Group (3 mentions) Alexa fluor 647, by Thermo Fisher Scientific (3 mentions) Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)

162 protocols using fluorsave reagent

Quantifying Autophagic Puncta in Cells

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The cells were transferred to slides (CultureWell Chambered Coverglass for cell culture, Invitrogen, C37000). Cells were synchronized in serum free media and treated with rapamycin and bafilomycin A1. After that they were fixed in 4% paraformaldehyde for 15 min and washed with phosphate-buffered saline (PBS) three times. The permeabilizing of cells were performed with 0,25% Triton-X (in PBS) for 10 min and then cells were washed three times in PBS for 5 min. The cells were blocked and incubated with antip62 (Cell Signaling, 7695S) according to the antibody protocol. Cells were washed with PBS and incubated with Alexa Fluor 488 conjugated anti-rabbit (Cell Signaling, 4412S) for 1 h. After washing the nuclei were staining with DAPI (1:10,000) for 5 min and then the cells were washed again. The slides were mounted with FluorSave Reagent (Millipore, 345,789) and observed under a fluorescence microscope (Nikon Eclipse Ts2R)^{23 (link)}. At each experimental step 4–6 different images were randomly photographed with similar settings. Total number of cells was counted by nuclei staining. The well demarcated, sharp p62 dots were counted in 80–100 cells using the program QuPath-0.2.1, which is freely available from https://github.com/qupath/qupath/releases/tag/v0.2.1.

Holczer M., Hajdú B., Lőrincz T., Szarka A., Bánhegyi G, & Kapuy O. (2020). Fine-tuning of AMPK–ULK1–mTORC1 regulatory triangle is crucial for autophagy oscillation. Scientific Reports, 10, 17803.

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Laminin Immunofluorescence and Cell Staining

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For laminin immunofluorescent staining, SA fibrous scaffolds were blocked with 3 v/v % goat serum (Sigma-Aldrich) for 30 min, followed with primary antibody, laminin (1:1000; Abcam) for 1 h, and secondary antibodies (Alexa Fluor dyes; Thermo Fisher Scientific) for 30 min. For cell culture experiments, cells with scaffolds were fixed in 4 v/v % paraformaldehyde (Sigma-Aldrich) for 20 min, permeabilized with 0.2 v/v % Triton X-100 (Sigma-Aldrich) for 15 min, and blocked with 3 v/v % goat serum for 45 min. Cells were then incubated for 1.5 h with primary antibodies, nestin (1:500; Millipore, U.K.), βIII-tubulin (1:1000; Sigma-Aldrich), and Ki67 (1:1000; Abcam), followed with DAPI (Sigma-Aldrich) and secondary antibodies for 45 min. The stained samples were mounted on slides with FluorSave Reagent (Millipore) and stored at 4 °C. Images of laminin immunofluorescent staining were acquired with an epifluorescent microscope (EVOS FL Cell Imaging System; Life Technologies, U.K.), whereas images of the cell experiments were acquired with a SP5MP/FLIM inverted confocal microscope (Leica, Germany) by sequential scanning. The thickness of the acquired sample sections was about 40 μm, and z stacks of typically 20 2 μm slices were imaged.

Hsu C.C., Serio A., Amdursky N., Besnard C, & Stevens M.M. (2018). Fabrication of Hemin-Doped Serum Albumin-Based Fibrous Scaffolds for Neural Tissue Engineering Applications. ACS Applied Materials & Interfaces, 10(6), 5305-5317.

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Immunostaining Assay for Acetylated Tubulin

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The immunostaining assay protocol was modified from the study by Wagner and Levine (58 (link)). In brief, the larvae were fixed at 20 or 24 hours after fertilization in MEM-FA. After washes in PBT, pretreatment in PBS with 0.1% Triton, washes in PBT, and incubation for 30 min in blocking solution at room temperature [10% Western blocking solution (Sigma-Aldrich) in PBT], the larvae were incubated at 4°C overnight with anti–acetylated tubulin antibody (1:500; Sigma-Aldrich, T7451) in blocking solution. After washes in PBT, the primary antibody was detected using goat anti-mouse antibody conjugated to Alexa Fluor 555 (1:1000; Thermo Fisher Scientific, A21424) diluted in blocking solution for 45 min at room temperature. The larvae were washed several times in PBT and mounted on slides using FluorSave Reagent (Millipore).

Lemaire L.A., Cao C., Yoon P.H., Long J, & Levine M. (2021). The hypothalamus predates the origin of vertebrates. Science Advances, 7(18), eabf7452.

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Electroporation and Imaging of Sea Urchin Embryos

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After fertilization, one cell-stage embryos were electroporated using 20 to 100 μg of each expression construct as described in Corbo et al, 1997^{10 (link)}.
The embryo were rise at 16, 18 or 21°C in ASW; fixed at the desired stage following the protocol described in Wagner and Levine 2012^{54 (link)}. The embryo were washed several times with 0.05% BSA in PBS before being mount using FluorSave™ Reagent (Millipore). Images were acquired with a Zeiss 880 confocal microscope with or without the Airyscan module, and a wide field Zeiss Axio Observer Z1/7 combined to the Apotome 2.0 module.
All electroporation were performed in duplicate or triplicate (see related figure legends). Between 18 and 610 embryos were recovered per condition. No specific randomization strategy was performed except the assignment of the fertilized eggs to the different conditions.
Live imaging was performed on a home-build two-photon microscope system. Embryos were anesthetized with 16mg/ml MS-222 in ASW (Sigma-Aldrich). They were placed in microwells casted in 1% agarose in ASW^{59 (link)} and the imaging was performed at 18 degree from LTBII to LTBIII stage. The images were assembled using Fiji^{60 (link)} and the final rendering obtained with Imaris (Bitplane, Switzerland).

Cao C., Lemaire L.A., Wang W., Yoon P.H., Choi Y.A., Parsons L.R., Matese J.C., Wang W., Levine M, & Chen K. (2019). Comprehensive single cell transcriptome lineages of a proto-vertebrate. Nature, 571(7765), 349-354.

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Quantification of DNA Damage Response

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Cells were plated in 35-mm glass bottomed culture dishes (Mattek), and were cultured overnight. The cells were then left untreated (DMSO) or treated with 10 μM Rucaparib or iRucaparib for 48 hrs before washing with PBS. Cells were fixed with 4% Paraformaldehyde for 15 min at RT and were washed three times with PBS. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min and were blocked with 1% BSA in PBS for 30 min. Fixed cells were incubated with a γH2AX antibody (1:200, Cell Signaling Technology, #9718) at 4℃ for overnight, washed three times with PBS for 5 min, and incubated with an Alexa Fluor 488 conjugated anti-rabbit antibody (1:500, Thermo Fisher Scientific, A-11008) for one hour at RT. Cells were washed three times with PBS for 5 min and stained with DAPI (1:1000, Thermo, #62248) for 2 min. Cells were washed with PBS and mounted with the FluorSave reagent (Millipore, #345789). Images were collected on a Zeiss LSM 880 Airyscan inverted confocal microscope.

Wang S., Han L., Han J., Li P., Ding Q., Zhang Q.J., Liu Z.P., Chen C, & Yu Y. (2019). Uncoupling of PARP1 Trapping and Inhibition Using Selective PARP1 Degradation. Nature chemical biology, 15(12), 1223-1231.

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Immunostaining of Paraffin-Embedded Tissue

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Cells were fixed in methanol previously cooled to −20°C and then washed with DPBS. Paraffin embedded sections, cut to 8 μm, were dewaxed and rehydrated through a series of alcohol. Cells/slides were blocked for 60 min at room temperature then incubated with primary over night at 4°C. Samples were then washed and incubated with secondary antibodies for 1 h at room temperature and were then washed and incubated in 1 μg/ml DAPI (Life Technologies). Coverslips were mounted onto Superfrost® plus (VWR) microscope slides using Fluorsave reagent (Millipore). Samples were imaged using an Invitrogen™ EVOS™ FL Auto cell imaging system at 60× magnification and a Nikon™ A1R™ confocal at 100× magnification. Unless stated, all immunostaining data was obtained from β-III tubulin/MAP2-positive cells.

Wallings R., Connor-Robson N, & Wade-Martins R. (2019). LRRK2 interacts with the vacuolar-type H+-ATPase pump a1 subunit to regulate lysosomal function. Human Molecular Genetics, 28(16), 2696-2710.

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Immunofluorescence Imaging of Cultured Cells

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Cells were seeded onto glass coverslips and treated as indicated. Cells then were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA. Slides were incubated with primary antibody overnight at 4°C, incubated with fluorescent-conjugated secondary antibody for 1 h at room temperature, and followed with DAPI incubation for 10 min at room temperature. Slides were then mounted with FluorSave Reagent (Millipore) and kept at 4°C in the dark. Images were captured with a Leica TCS SPE microscope, 63× objective, and oil immersion.

Deng Z., Chong Z., Law C.S., Mukai K., Ho F.O., Martinu T., Backes B.J., Eckalbar W.L., Taguchi T, & Shum A.K. (2020). A defect in COPI-mediated transport of STING causes immune dysregulation in COPA syndrome. The Journal of Experimental Medicine, 217(11), e20201045.

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Immunofluorescence Localization of RLIM in mESCs

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For localisation studies, mESCs were plated in 0.1% gelatin (v/v) coated coverslips and fixed in 4% PFA in PBS for 20 min at room temperature (RT). Cells were permeabilised with a 0.5% Triton X-100 in PBS solution for 5 min at RT and then blocked with 1% Fish gelatin (w/v) in PBS solution for 30 min at RT. RLIM primary antibody (Novus Biologicals) was diluted 1:200 in blocking solution and added to cells for 2 h at RT. Anti-mouse Alexa-488 (Thermo Fisher Scientific) was used as a secondary antibody at 1:500 in blocking solution for 1 h at RT. Actin Red 555 reagent (Thermo Fisher Scientific, one drop per ml of blocking solution) was added together with secondary antibody for actin staining. Hoechst was added at 1:10,000 dilution in PBS for 5 min at RT as nuclear marker. Coverslips were mounted in glass slides using Fluorsave reagent (Millipore). Digital images were acquired in a Zeiss 710 confocal microscope and analysed and processed using ImageJ (NIH), Photoshop CC and Illustrator CC (Adobe).

Bustos F., Espejo-Serrano C., Segarra-Fas A., Toth R., Eaton A.J., Kernohan K.D., Wilson M.J., Riley L.G, & Findlay G.M. (2021). A novel RLIM/RNF12 variant disrupts protein stability and function to cause severe Tonne–Kalscheuer syndrome. Scientific Reports, 11, 9560.

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Immunofluorescence Assay for LC3A/B

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The cells were transferred to a chamber slide (Nunc™ Lab-Tek™ II Chamber Slide™ System, Thermo Fisher Scientific Inc., 154453PK). After silencing and treatment, the cells were washed in phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde for 10 min and washed again with ice-cold PBS three times. The fixed cells were permeabilized with 0.1% Triton-X (in PBS) for 10 min and then washed in PBS three times for 5 min. After permeabilization, the cells were blocked with PBS with 0.1% Tween-20 containing and 3% bovine serum albumin for 30 min and incubated overnight with antiLC3A/B (Cell Signaling, 4108S) in 1% bovine serum albumin containing PBS at 4 °C. Cells were washed with cold PBS and incubated with Alexa Fluor 488 conjugated anti-rabbit (Cell Signaling, 4412S) for 1 h. Cells were washed in PBS and treated with DAPI (1:10000) for 15 min and washed again twice for 3 min. The slides were mounted with FluorSave Reagent (Millipore, 345789, Burlington, MA, USA) and observed under a fluorescence microscope (Nikon Eclipse Ts2R, Minato, Tokyo, Japan).

Holczer M., Hajdú B., Lőrincz T., Szarka A., Bánhegyi G, & Kapuy O. (2019). A Double Negative Feedback Loop between mTORC1 and AMPK Kinases Guarantees Precise Autophagy Induction upon Cellular Stress. International Journal of Molecular Sciences, 20(22), 5543.

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Cytotoxicity and ROS Evaluation of SNP

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Sodium nitroprusside dihydrate (Na₂[Fe(CN)₅NO]·2H₂O, SNP, ≥99%) (SNP), silver nitrate (AgNO₃), and anhydrous methanol (99.8%) were purchased from Sigma-Aldrich (St. Louis, USA). A Millipore Milli-Q Biocell A10 water purifying system was used to prepare ultrapure water. A2780, A2780cis, U-87 MG (Sigma-Aldrich, St. Louis, MO, USA), MDA-MB-231 (Cell Biolabs, CA, USA) SK-OV-3, MCF-7, MRC-5, W1-38 (ATCC, Manassas, VA, USA) cells were cultured according to the manufacturers’ instruction. LysoTracker Green DND-26, Hoechst 33342, and rhodamine B were purchased from (ThermoFisher Scientific, Waltham, MA, USA). CellTiter-Glo® from (Promega, Madison, WI, USA), PE-Annexin V Apoptosis Detection Kit from (Becton-Dickinson Franklin Lakes, NJ, USA). ROS-Glo™ H₂O₂ luminescence assay from (Promega, Madison, WI, USA), DAX-J2 PON Green Kit was purchased from (AAT Bioquest, Sunnyvale, CA, USA), FluorSave™ reagent (Millipore, Burlington, MA, USA). qPCR Primers from (Integrated DNA Technologies, Bologna, Italy).

Asif K., Adeel M., Rahman M.M., Sfriso A.A., Bartoletti M., Canzonieri V., Rizzolio F, & Caligiuri I. (2023). Silver nitroprusside as an efficient chemodynamic therapeutic agent and a peroxynitrite nanogenerator for targeted cancer therapies. Journal of Advanced Research, 56, 43-56.

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