After 16-hour treatment, primary patient mononuclear cells were incubated with 7-AAD (SigmaAldrich) and anti-human CD34 antibody conjugated with Alexa Fluor488 (Biolegend, Cat#343518) for 20 minutes. Slides were then mounted using DAPI (4’,6-diamidino-2-phenylindole.) Fluoromount-G (Southern Biotech, Birmingham, AL). PDX bone marrow samples were fixed in 4% formaldehyde for 2 minutes and then blocked with PBX containing 5% BSA. Anti-human CD45-PE/Cy7 (BD Pharmingen, Cat#557748) was employed for immunofluorescent staining. Slides were then mounted using DAPI (4’,6-diamidino-2-phenylindole.) Fluoromount-G (Southern Biotech, Birmingham, AL). Images were captured using an Olympus IX71 Inverted System Microscope with a DP73; 17MP Color Camera.
Fluoromount g
Manufactured by Southern Biotech
Sourced in United States, United Kingdom, Germany, Sweden, Japan, Panama, France, Colombia
Fluoromount-G is a water-soluble, synthetic, non-glycerin-based mounting medium designed for fluorescence microscopy. It is formulated to preserve the fluorescence of tagged samples and provide a durable, long-lasting mounting solution.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (102 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (68 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (65 mentions)
Hoechst 33342, by Thermo Fisher Scientific (49 mentions)
Lsm 710 confocal microscope, by Zeiss (46 mentions)
Triton x 100, by Merck Group (39 mentions)
Lsm 780 confocal microscope, by Zeiss (37 mentions)
Lsm 700 confocal microscope, by Zeiss (30 mentions)
Lsm 710, by Zeiss (29 mentions)
Lsm 700, by Zeiss (25 mentions)
Lsm 880 confocal microscope, by Zeiss (22 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (20 mentions)
Hoechst, by Thermo Fisher Scientific (19 mentions)
Paraformaldehyde (pfa), by Merck Group (19 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (19 mentions)
Cryostat, by Leica camera (18 mentions)
Alexa 488, by Thermo Fisher Scientific (18 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (18 mentions)
Lsm 780, by Zeiss (16 mentions)
Zen software, by Zeiss (15 mentions)
1 475 protocols using fluoromount g
1
Characterization of Primary Patient Mononuclear Cells
2
Visualizing Drosophila Ovaries and Hemocytes
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A Leica MZ FLIII fluorescence stereomicroscope associated with a Panasonic DMC-G2 camera was used to visualize whole flies anesthetized with CO2. Confocal images of ovaries were produced in a Zeiss LSM 780 microscope. The following dyes were used: DAPI (1:1000 dilution, Sigma-Aldrich), and Phalloidin-TRITC (1:1000 dilution, Sigma-Aldrich). The samples were mounted in Fluoromount-G (SouthernBiotech). To retrieve the hemocytes, flies were anesthetized, preinjected with small amount of Schneider’s medium (Sigma-Aldrich) containing the anti-coagulant phenylthiourea (PTU) and incubated on ice for 5 min. Thereafter cells were collected on a glass slide with 25 μl of Schneider’s medium with PTU and incubated in a wet chamber for 30 min to allow them to adhere. For fixation 4 % paraformaldehyde in PBS was used for 10 min. After staining with DAPI (1:1000 dilution, Sigma-Aldrich), samples were mounted in Fluoromount-G (SouthernBiotech) and observed under a Zeiss Axioplan 2 microscope coupled to a Hamamatsu ORCA-ER camera (C4742-95). The ImageJ program was used to measure the intensity of GFP. Fluorescence values were recalculated to Corrected total fluorescence (CTF) according to [170 (link)].
3
Pluripotency and Differentiation Characterization of Neural Progenitor Cells
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Pluripotency of neural progenitor cells was proven by staining for Sox2, nestin, and Pax6. Specimens were incubated with primary antibodies overnight at 4 °C and with secondary antibodies for 1 h at room temperature in 1% BSA/PBS. Subsequently, cells were washed three times with PBS and mounted with Fluoromount-G (SouthernBiotech, Birmingham, UK). Differentiated cells were stained for the neuronal marker βIII-tubulin and the glia cell marker GFAP. Therefore, cells were permeabilized using 0.1% Triton X-100 for 5 min on ice. Cells were incubated with primary antibodies overnight at 4 °C in 1% BSA/PBS, followed by three washing steps with PBS. Secondary antibodies were added for 1 h at room temperature in 1% BSA/PBS. After washing with PBS, cells were stained with DAPI (5 min, 250 ng/mL), washed three times, and mounted with Fluoromount-G® (SouthernBiotech, Birmingham, UK). Pictures were taken with a Biozero 8000 microscope system (Keyence, Hamburg, Germany). Used primary and secondary antibodies are listed in Table S1 .
4
Immunostaining Protocols for Mouse Microglia and Vasculature
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For mouse microglia immunostaining, eyes were enucleated and fixed in paraformaldehyde 4% for 1 hour. Subsequently, retinas and choroids were isolated, permeabilized, blocked and incubated with anti-Iba1 antibody (1/1000 dilution) (Abcam Ab178846) overnight, at room temperature. The next day, samples were washed, incubated with secondary antibody (1/200 dilution) (goat anti-rabbit AlexaFluor 595, Invitrogen A11012, USA) for 2 hours and washed again. Retinas and choroids were flat-mounted with Fluoromount-G (SouthernBiotech, The Netherlands) on glass-slides for microscopy imaging.
For mouse vascular immunostaining, eyes were enucleated and fixed in ethanol 70 % for 1 hour. Subsequently, choroids were isolated, permeabilized, blocked and incubated with anti-CD31 antibody (1/150 dilution) (Pharmingen 553370) overnight, at room temperature. The next day, samples were washed, incubated with secondary antibody (1/200 dilution) (goat anti-rat AlexaFluor 488, Invitrogen A11006) for 2 hours and washed again. Choroids were flat-mounted with Fluoromount-G (SouthernBiotech) on glass-slides for microscopy imaging.
For mouse vascular immunostaining, eyes were enucleated and fixed in ethanol 70 % for 1 hour. Subsequently, choroids were isolated, permeabilized, blocked and incubated with anti-CD31 antibody (1/150 dilution) (Pharmingen 553370) overnight, at room temperature. The next day, samples were washed, incubated with secondary antibody (1/200 dilution) (goat anti-rat AlexaFluor 488, Invitrogen A11006) for 2 hours and washed again. Choroids were flat-mounted with Fluoromount-G (SouthernBiotech) on glass-slides for microscopy imaging.
5
Immunofluorescence Assay for NF-κB Translocation
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RBMVECs were seeded and cultured on gelatin-coated glass coverslips until confluent. Confluent monolayers were then transfected with Poldip2 or control siRNA as described above. Forty-eight hours post-transfection, cells were stimulated with 1 μg/mL LPS for 1 h. After 1 h, cells were washed twice in HBSS and fixed in 3.7% paraformaldehyde diluted in HBSS for 10 min at room temperature. Cells were then washed three times in PBS and permeabilized in PBS containing 0.2% Triton X-100 for 5 min followed by three washes in PBS for 2 min each. Blocking was performed in 0.5% BSA in PBS for 30 min. Anti-NF-κΒ p65 monoclonal rabbit Ab (Cell Signaling; Cat No. D14E12) was diluted 1:500 in blocking buffer and coverslips were incubated overnight at 4 °C with gentle rocking. The following day, coverslips were rinsed in blocking buffer (three washes × 5 min each) before incubation with secondary (Alexa488) antibody (1:500 dilution, Invitrogen) and DAPI (1:1000 dilution) for 1 h. Cells were then rinsed three times in PBS for 5 min each. Finally, coverslips were mounted on glass slides using Fluoromount-G (SouthernBiotech; Cat No. 0100-01).
6
Quantifying mT/mG and Leptin-Luciferase Adipocytes
7
Immunostaining protocol for cryosections
8
Neuromuscular Junction Staining Protocol
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TVA, ETA, FDB, and lumbrical muscles of both the hands and feet were dissected (Murray et al., 2014; Sleigh et al., 2014a; Tarpey et al., 2018) and stained as previously described (Sleigh et al., 2017 (Sleigh et al., , 2014b)) . Briefly, muscles were excised and fixed for 10 min in 4% (w/v) paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA) in phosphate buffered saline (PBS), before being permeabilised for 30 min in 2% (v/v) Triton X-100 in PBS and blocked for 30 min in 4% (w/v) bovine serum albumin and 1% (v/v) Triton X-100 in PBS. Muscles were then probed overnight at 4˚C in blocking solution with 1/250 mouse anti-neurofilament (2H3, Developmental Studies Hybridoma Bank [DSHB], Iowa City, IA, supernatant) and 1/25 mouse pan anti-synaptic vesicle 2 (SV2, DSHB, supernatant) antibodies to co-visualise axons and presynaptic nerve terminals, respectively. The following day muscles were washed several times with PBS before being incubated with 1/1,000 Alexa Fluor 488 donkey anti-mouse IgG secondary antibody (Life Technologies, Carlsbad, CA, A-21202) and 1/1,000 Alexa Fluor 555 α-bungarotoxin (α-BTX, Life Technologies, B35451) in PBS for 2 h. Muscles were washed with PBS and then mounted in Fluoromount-G (Southern Biotech, Birmingham, AL, 0100-01) on microscope slides for imaging. All five muscles from each mouse were processed in parallel.
9
Cytospin-based Visualization of Infected Cells
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Freshly isolated bone marrow cells from infected mice were collected on slides using a cytospin centrifuge (Hettich, Tuttlingen, Germany) at 800rpm for 5 min. The smears were air dried and stained with modified Wright-Giemsa stain (Protocol Hema3; Thermo Fisher Scientific) as per manufacturer’s instructions. Slides were mounted using Fluoromount-G (SouthernBiotech) and coverslips sealed with nail polish. Analyses of infected cells were performed using a Nikon Eclipse E800 microscope (Nikon) with a 100x oil immersion lens and images were captured with a digital camera (COOLPIX 990; Nikon).
10
Immunofluorescence Staining and Imaging
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Fixed cultures were permeabilized for 1 h in blocking solution [3% BSA (w/v), 10% normal goat serum (v/v), 0.2 M glycine in PBS, 0.1% Triton-X100 (v/v)] (Kontou et al., 2021 (link)). Coverslips were incubated with primary antibodies diluted in blocking solution for 1 h at room temperature. After a brief wash with PBS, the coverslips were incubated with fluorophore-conjugated secondary antibodies diluted in blocking solution for 1 h at room temperature. The coverslips were then washed in PBS, briefly dipped in DAPI ready-made solution (Sigma-Aldrich MBD0015), and mounted onto microscope slides with Fluoromount-G (SouthernBiotech 0100-01). The coverslips were imaged using a Nikon A1 confocal microscope (Nikon Instruments, Melville, NY, USA) using a 60× oil immersion objective lens. Laser settings were manually assigned for each fluorescent channel and images were acquired at the scan size of 1,024 × 1,024. Settings were kept the same between imaging sessions and between genotypes.
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