Briefly, slides were washed in PBS (10 mM), then glycine (0.1 M, diluted in PBS 10 M) and then in PBS-T (0.1% Triton X-100; Sigma, St. Louis, MO, USA). Sections were incubated with bovine serum albumin Fraction V (Sigma, St. Louis, MO, USA) in PBS-T and then with anti-parvalbumin primary antibody (1:2000; Millipore, Billerica, MA, USA) overnight. Afterwards, slides were washed in PBS, and incubated with secondary antibody conjugated to fluorophore (Alexa-594; 1:1000; Invitrogen, Carlsbad, CA, USA) for 2 h. Slides were then washed in PBS and mounted with Fluoroshield medium with DAPI (Sigma, St. Louis, MO, USA).
Fluoroshield medium with dapi
Manufactured by Merck Group
Sourced in United States
Fluoroshield medium with DAPI is a mounting medium designed for the preservation and visualization of fluorescently labeled samples. It contains the fluorescent dye DAPI, which stains DNA and facilitates the identification of cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (9 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin fraction 5, by Merck Group (1 mentions)
Pbs t, by Merck Group (1 mentions)
C3867, by Merck Group (1 mentions)
Ab10062, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Zen10, by Zeiss (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Chicago sky blue, by Merck Group (1 mentions)
Dq gelatin, by Thermo Fisher Scientific (1 mentions)
Mn1000, by Thermo Fisher Scientific (1 mentions)
Rat tail collagen type 1, by Merck Group (1 mentions)
Anti iba1 antibody, by Fujifilm (1 mentions)
6 protocols using fluoroshield medium with dapi
1
Immunofluorescent Labeling of Parvalbumin
2
Visualizing hACE2 Expression in HEK293T Cells
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HEK293T/17-hACE2 cells were plated on cover glass pre-coated with rat-tail collagen, type I (C3867, Sigma-Aldrich) and cultivated for 24 h in DMEM with 10% FCS. Cells were fixed with 4% paraformaldehyde and labelled with anti-human ACE2 antibody at 10 µg/ml (cat. # 600-401-X59; Rockland Immunochemicals), followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (Alexa Fluor 488; #A-11008; Invitrogen). The samples were mounted in Fluoroshield™ medium with DAPI (Sigma-Aldrich). Images were captured by LSM 710 confocal microscope (Zeiss, Jena, Germany).
3
Immunostaining of Microglia and Astrocytes
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Cells were fixed with 4% paraformaldehyde (PFA) for 10 min at 37°C, blocked and permeabilized with 5% BSA and PBS 0.1% Triton-X100 for 60 min at room temperature (RT), were washed three times and incubated O/N with primary antibody anti-Iba1 (1:500, rabbit polyclonal antibody; Wako Pure Chemical Industries, Osaka, Japan) and anti-GFAP (1:200, mouse monoclonal antibody, ab 10,062, Abcam) in 5% BSA 0.1% Triton-X100 PBS. Slides were washed three times and incubated with the secondary fluorescent antibodies, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) and Alexa Fluor 546 goat anti-mouse IgG (Invitrogen), both diluted 1:2000 in 5% BSA 0.1% Triton-X100 PBS, for 2 h at room temperature (RT) in the dark. The slides were mounted using Fluoroshield medium with DAPI (Sigma). Labelled cells were analysed with fluorescence microscope Zeiss LSM710. The microscope pictures were processed with Zen10 software (Zeiss).
4
Measuring Gelatinase Activity in Cardiac Cryosections
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Non-fixed hearts were snapfrozen on dry ice and stored until further use. 10 mm cryosections were acquired from the three-valve area and air dried for 1e2 h before storage at À80 C. Cryosections were washed in reaction buffer (150 mM NaCl, 5 mM CaCl 2 , 50 mM Tris-HCl, pH ¼ 7.6) and incubated in reaction buffer containing 30 mg/mL DQ-Gelatin (Molecular probes) and 20 mM MMP2inhibitor OA-Hycis-9-Octadeconoyl-N-hydroxylamide (Merck Millipore) overnight at 37 C. 1 mM 1,10-phenantrolin is an MMP inhibitor and thus served as a control. After incubation, the sections were washed in reaction buffer and subsequently incubated in 0.5% Chicago Sky Blue (Sigma) for 5 min to dampen autofluorescence. Finally, the slides were mounted in Fluoroshield medium with DAPI (Sigma) and imaged using the Leica Image analysis system (Leica Ltd).
5
Studying Tau Pathology in Rodent Neurons
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Rodent cortico-hippocampal neurons were cultured on cover glass pre-coated with Poly-D-lysine at a density of 1 × 106 cells per milliliter and cultivated for 48 h. Neurons were treated with fluorescently labelled (Alexa Fluor 488 or Fluor 594) sarkosyl-insoluble AD tau (2p, 100 nM) only or in combination with control and DC8E8/AX004 antibody (1 μM) and cultivated for 20 h in conditioned Neurobasal media at 37 °C, 5% CO2. Next day neurons were washed with pre-warmed PBS and mild trypsin (0,06% trypsin-EDTA, 3 min), fixed with 4% PFA-PHEM, pH 6.9 (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, PFA) for 12 min. Neurons were permeabilized with 0.1% Triton X100 in PBS (PBS-T) and blocked with 5% BSA in TBS-T. The cells were then incubated with anti-heparan sulfate antibody (10E4, AMSBIO 1:100) for 1 h or, in a different experiment with HT7 antibody (epitope against human tau, MN1000, Thermo Fisher Scientific 1:500), washed and incubated with secondary antibody goat anti-mouse Alexa Fluor 488 (Invitrogen). The samples were mounted in FluoroshieldTM medium with DAPI (Sigma-Aldrich). Images were captured by LSM 710 confocal microscope (Zeiss, Jena, Germany).
6
Microglia Characterization and Tau Uptake Analysis
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Microglia cells were plated on cover glass pre-coated with rat-tail collagen, type I (Sigma-Aldrich, St. Louis, Missouri, United States) and cultivated for 24 h in DMEM with 10% FCS. For basic analysis of microglia, cells were washed with PBS, fixed with 4% PFA-PHEM, pH 6.9 (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, PFA) for 12 min, permeabilized with 0.1% Triton X-100 in PBS (PBS-T) for 3 min followed by blocking with 5% BSA in TBS-T for 1 h. The cells were then incubated with anti-Iba1 antibody (WAKO, 1:1000) for 1 h, rinsed and incubated with secondary antibody goat anti-mouse AlexaFluor 488 (Invitrogen). For tau uptake analysis, cells were incubated with tau-488 alone and in combination with antibodies DC8E8 or Rab51 for 20 min. Then microglia were washed with PBS, mildly trypsinized (0.06% trypsin-EDTA) for 3 min and three times washed with PBS followed by fixation with 4% PFA-PHEM, pH 6.9 for 12 min. The samples were mounted in FluoroshieldTM medium with DAPI (Sigma-Aldrich). Images were captured by LSM 710 confocal microscope (Zeiss, Jena, Germany).
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