Cells were washed in PBS before fixation with 4% paraformaldehyde for 15 min and then permeabilized with 0.2% Triton-X 100 for 10 min at room temperature (RT). After blocking with 3% BSA for 1 hr, cells were incubated with primary antibody at the recommended dilution, followed by Alexa-Fluor 594- or 488-conjugated secondary antibodies (Invitrogen, Thermo Fisher Scientific) at a 1:100 dilution for 1 hr. Nuclei were labeled with Hoechst-33342 (Thermo Fisher Scientific) before cells were mounted onto a microscope slide using Mowiol® 4-88. Finally, IF-staining images were captured with a Zeiss Axio Observer microscope. The primary antibodies used in this study are listed in Table S2 .
Mowiol 4 88
Manufactured by Thermo Fisher Scientific
Mowiol® 4–88 is a water-soluble polyvinyl alcohol (PVOH) resin. It is commonly used as a binder, emulsifier, and film-forming agent in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Polysine coated slides, by Thermo Fisher Scientific (2 mentions)
Anti tlr2 blocking antibody t2, by InvivoGen (2 mentions)
Anti mouse c5b 9, by Santa Cruz Biotechnology (2 mentions)
Phallodin, by Thermo Fisher Scientific (2 mentions)
Lsm 700 t pmt scanning, by Zeiss (2 mentions)
Axio observer z1 inverted microscope, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 or 488 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dabco, by Thermo Fisher Scientific (1 mentions)
Mouse anti ki 67, by Leica (1 mentions)
Goat anti rabbit igg af488, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
5 protocols using mowiol 4 88
1
Immunofluorescence Staining Protocol
2
Polarized ARPE-19 Cells: Serum-Induced MAC Formation
3
Quantifying Apoptosis and Proliferation in OCT Biofilms
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OCT embedded biofilm wounds, sectioned at 10 μm, were blocked in appropriate serum and incubated in primary antibodies O/N at 4°C. The antibodies used were the early apoptosis marker, goat anti-caspase 3 (R&D Systems), and the cell-proliferation marker, mouse anti-Ki67 (Novocastra). Alexa Fluor conjugated secondary antibodies (Thermo Fisher Scientific) were used to detect antibody binding. Sections were counterstained in DAPI and mounted in Mowiol 4-88 with DABCO (Thermo Fisher Scientific). Fluorescent images were taken as above using the DAPI, FITC and TEXAS RED filters.
4
Polarized ARPE-19 Cells: Serum-Induced MAC Formation
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Polarized ARPE-19 cells grown on transwell filters were maintained in serum free DMEM F-12 Ham for 48 hours. Cells were stimulated with 10% Normal Human Serum or heat inactivated Normal Human Serum (Hi) (56°C 30 minutes) either alone or with human serum albumin (HSA) or CEP-HSA for 24 hours. Where indicated cells were pre-treated for 1 hour with anti-TLR2 blocking antibody (T2.5 Invivogen), IgG control (0.1 µg/ml) or Mal peptide inhibitor (Calbiochem). Supernatants were harvested and assessed for soluble MAC formation by ELISA (Abbexa). Transwell inserts were fixed with 4% Paraformaldehyde for 10 minutes at room temperature, blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and incubated with anti-mouse-C5b-9 1:25 (Santa cruz) overnight at 4°C. Transwells were washed 3 times in PBS and incubated with goat anti-mouse 647 1:500 (Invitrogen) and Phallodin 1:500 (Invitrogen) for 2 hours at room temperature. Cells were counted stained with Hoechst. Transwells inserts were carefully cut with a sterile blade and mounted on to polysine coated slides (Thermo Scientific) using Mowiol® 4–88. Staining was analyzed using a confocal laser scanning microscope Axio Observer Z1 Inverted Microscope equipped with a Zeiss LSM 700 T-PMT scanning unit and a 40x plan.
5
Localization of TFEB and TFE3 in iBMDMs
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Immortalized bone marrow derived macrophages (iBMDMs) (49 ) were cultured in complete DMEM. Cells were passaged 1:10 every 3 days. To assess the subcellular localization of TFEB or TFE3 in iBMDM cells, 5×105 cells were seeded into 12 well plates on top of coverslips in cDMEM. Swollen heat killed conidia (SHKC) were prepared as described above and 5×106 SHKC were added to the appropriate wells (MOI = 10). Following stimulation, cells were fixed using 4% paraformaldehyde (PFA) in PBS and permeabilized with PBS containing 0.25% Triton X-100. Cells were then incubated with rabbit α-TFEB (1:1000) (ThermoFisher cat#: 50-156-5746) or rabbit α-TFE3 (1:1000) (ThermoFisher cat#: HPA023881) followed by incubation with goat anti-rabbit IgG AF488 (ThermoFisher cat#: A-11008). Finally, cells were counterstained with 1 µg/mL DAPI, mounted on slides with Mowiol 4–88 (ThermoFisher cat#: 47-590-4100GM), and imaged using a Leica TCS SP5 Confocal Microscope. Nuclear TFEB or TFE3 were calculated by outlining DAPI-positive structures and calculating the average TFEB or TFE3 staining intensity in each region, using Fiji (50 (link)).
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