For immunofluorescence microscopy, cells were either fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature or with methanol for 5 min at −20°C. Antibodies were diluted in PBS with 0.1% (v/v) Triton-X100 and 10% (v/v) horse serum. The cells were washed in PBS with 0.1% (v/v) Triton-X100 and incubated for 1 h with primary antibodies. The cells were washed again in PBS with 0.1% (v/v) Triton-X100 and incubated with appropriate secondary antibodies for another 1 h. The cells were washed again first in PBS with 0.1% (v/v) Triton-X100, then PBS, and finally in H2O. In the end, coverslips were mounted onto glass slides using Aqua-poly/Mount mounting medium (Polysciences). Samples were analyzed using widefield fluorescence microscopes (Leica DMRA2 or Zeiss AxioImager D2) or a confocal laser scanning microscope (Zeiss LSM 710 META). Unless otherwise indicated, images were obtained by confocal microscopy and processed using Zeiss LSM Image Browser and Adobe Photoshop software or Fiji (Schindelin et al., 2012 (link)).
Aqua poly mount mounting medium
Manufactured by Polysciences
Sourced in United States
Aqua Poly/Mount is a water-soluble mounting medium. It is designed for use in microscopy applications to mount and preserve biological samples on slides.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Tcs sp2 aobs confocal microscope, by Leica (2 mentions)
Normal donkey serum (nds), by Merck Group (2 mentions)
Alexa fluor 488 conjugated donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti ki67 primary antibody, by Thermo Fisher Scientific (2 mentions)
Lsm 710 meta, by Zeiss (1 mentions)
Axio imager d2, by Zeiss (1 mentions)
Lsm image browser, by Adobe (1 mentions)
Eclipse te2000 u inverted microscope, by Nikon (1 mentions)
Digital sight ds fi1 camera, by Nikon (1 mentions)
Ba d5, by Developmental Studies Hybridoma Bank (1 mentions)
Bf f3, by Developmental Studies Hybridoma Bank (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Mgso4, by Merck Group (1 mentions)
D glucose, by Worthington (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Rat anti brdu, by Abcam (1 mentions)
18 protocols using aqua poly mount mounting medium
1
Immunofluorescence Microscopy Protocol
2
Muscle Fiber Typing by Immunofluorescence
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For fiber typing, 8 μm thick transverse sections of soleus or TA muscles were washed with PBS and blocked with 2% bovine serum albumin (BSA) for 45 minutes followed by incubation with monoclonal antibodies against type I, IIa, and IIb MyHC isoforms using clone BA‐D5, SC‐7, and BF‐F3, respectively (Developmental Studies Hybridoma Bank) for 1 hour. The slides were washed and then incubated with goat anti‐mouse IgG2b conjugated with Alexa‐350, goat anti‐mouse IgG1 conjugate with Alexa‐568 and goat anti‐mouse IgM conjugated with Alexa‐488 secondary antibodies. The slides were then mounted using Aqua‐Poly/Mount mounting medium (Polysciences, Inc, Catalog No.18606). All images were captured using Eclipse TE 2000‐U Nikon inverted microscope mounted with Digital Sight DS‐Fi1 camera at room temperature.
3
Quantifying Amyloid Plaque Deposition
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To quantify amyloid plaques, transcranial perfusion with 4% paraformaldehyde diluted in PBS was performed 60 days after injection. For further immunohistochemical staining, fixed 100 µm thick brain slices were used. During the selection of conditions, the standard protocol for immunohistochemical staining was improved using the HCl antigen retrieval method, as preliminary data showed that the best visualization of amyloid accumulations in brain tissue occurs when pre-enhanced staining by antigen demasking. After 20 min of incubation with 2 N HCl, slices were blocked with 5% BSA diluted in PBS with 0.25 % Triton X-100. Slices were then incubated overnight in a 6E10 primary monoclonal antibody solution (1:1000 dilution, Biolegend Cat# 803001), solution in PBS with 5 % BSA and 0.125 % Triton X-100. After, slices were incubated for two hours with secondary antibodies Alexa Fluor-488 (anti-mouse, Invitrogen, cat. no. A11001) diluted 1:1000 in PBS with 5% BSA and 0.125 % Triton X-100. After that, they were placed under coverslips using Aqua Poly/Mount mounting medium (Polysciences, Inc., USA) and the amyloid plaques were analyzed using a confocal microscope. The percentage of the surface occupied by plaques from the total scanned area was used as parameter to assess the accumulation of β amyloid in the brain using the freely available Icy software.
4
Isolation and Culture of Mouse Embryonic Cortical Neurons
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Cortices from E15.5 CD1 mouse embryos were dissected in cold PBS supplemented with BSA (3 mg/mL), MgSO4 (1 mM, Sigma), and D-glucose (30 mM, Sigma). Cortices were dissociated in Neurobasal media containing papain (20 U/mL, Worthington) and DNase I (100 μg/mL, Sigma) for 20 min at 37 °C, washed 5 min with Neurobasal media containing Ovomucoïde (15 mg/mL, Worthington), and manually triturated in OptiMeM supplemented with d -Glucose (20 mM). Cells were then plated at 2 × 105 cells per 24-well plate coated with poly-d -lysine (1 mg/mL, Sigma) overnight at 4 °C and cultured for 2–5 days in Neurobasal medium supplemented with B27 (1×), l -glutamine (2 mM) and penicillin (5 units/mL)–streptomycin (50 mg/mL). To transfect cultured neurons, we performed magnetofection at DIV0 (for analysis at DIV2) or DIV2 (for analysis at DIV5) using NeuroMag (OZ Bioscience) according to the manufacturer’s protocol. Cells were fixed at DIV2 or DIV5 for 15 min at room temperature in 4% PFA, 4% sucrose in PBS, and incubated for 1 h in 0.1% Triton X-100, 5% NDS in PBS. Primary antibodies were incubated overnight at 4 °C and secondary antibodies were incubated for 1 h at room temperature (see Supplementary Table 1 for antibodies). DNA was stained using DAPI (1/1000). Slides were air-dried and mounded in Aquapolymount mounting medium (Polysciences Inc).
5
Immunohistochemical Staining Protocol
6
Quantifying Enteric Nervous System Lineages
7
Immunofluorescence and Proximity Ligation Assay of Mouse Aortic VSMC
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Cells grown on coverslips were fixed by addition of 10% formaldehyde to the final concentration of 2%, permeabilized in 0.1% Triton X-100 for 10 min and blocked with 3% (w/v) BSA/PBS at 4°C overnight. Cells were labeled with primary and corresponding Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibody (Invitrogen) for 1 h at room temperature. Cells were then mounted with Aqua-Poly-Mount mounting medium (Polysciences) and analyzed on a Leica TCS-SP2 AOBS confocal microscope. For immunostaining of mice Aortic VSMC, cells were incubated with 5% mouse serum in PBS followed by 1 h incubation with 5% normal goat serum.
Duolink In Situ Proximity Ligation Assay (PLA) Probes (anti-rabbit PLA probe PLUS, anti-mouse PLA probe MINUS, and Duolink In Situ Detection Reagent Green) were purchased from Sigma-Aldrich and used accordingly to the manufacturer’s instructions. The number of PLA signals was quantified using ImageJ software.
Duolink In Situ Proximity Ligation Assay (PLA) Probes (anti-rabbit PLA probe PLUS, anti-mouse PLA probe MINUS, and Duolink In Situ Detection Reagent Green) were purchased from Sigma-Aldrich and used accordingly to the manufacturer’s instructions. The number of PLA signals was quantified using ImageJ software.
8
Immunofluorescence Analysis of γ-Radiation Damage
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Immunofluorescence was performed on cells grown overnight on coverslips and then treated with γ-radiation; they were then fixed with 2% formaldehyde at the required time points, permeabilized with 0.1% Triton X-100 and blocked with 3% BSA in PBS at 4 °C overnight. Cells were labeled with primary and corresponding Alexa Fluor® 488- or Alexa Fluor® 594-conjugated secondary antibody (Invitrogen) for 1 h at room temperature. DRAQ5™ (Biostatus) was used for nuclear staining. Cells were then mounted with Aqua-Poly-Mount mounting medium (Polysciences) and analyzed on a Leica TCS-SP2 AOBS confocal microscope. Antibody for 8-OHdG (bs 1278R) was from Bioss.
9
Quantifying Lung Metastasis in Mice
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Lungs is the common analyzed site of metastasis after subcutaneous injection of tumor cells in mice [27 (link)]. Cross sections of isolated mice lungs were obtained and then fixed in 4% paraformaldehyde (Panreac, Barcelona, Spain), washed in PBS, permeabilized with 0.1% Triton X-100 (Triton®X-100, Peroxide Free, Panreac) and washed in PBS. To block the non-specific binding, sections were treated with 10% normal donkey serum (Sigmaaldrich) for 1 h, washed in PBS and counterstained with DAPI Sigmaaldrich, 1:10,000. Samples were embedded in Aqua Poly Mount mounting medium (Polysciences, Warrington, PA, USA). Images of lung metastases were acquired using confocal laser scanning microscopy system Zeiss (LSM 780) and ZEN 2010 software. DAPI and GFP fluorescence were sequentially excited using lasers with 405 and 488 wave lengths, respectively. The number of GFP+ cells per image view was calculated using the ImageJ software (NIH).
10
Immunofluorescence Staining of SEPT7
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Infected or uninfected cells were washed 3× with PBS pH 7.4 and fixed 15 min in 4% paraformaldehyde (in PBS) at RT. Fixed cells were washed 3× with PBS pH 7.4 and subsequently permeabilized 5 min with 0.1% Triton X-100 (in PBS). Cells were then washed 3–6× in PBS and incubated 1 h 30 min with primary anti-SEPT7 antibody diluted in PBS supplemented with 0.1% Triton X-100 and 1% bovine serum albumin. Cells were then washed 3–6× in PBS and incubated 45 min with Alexa-555-conjugated anti-rabbit secondary antibody diluted 0.1% Triton X-100 (in PBS). Cells were then washed 3–6× in PBS and incubated 40 min with a solution of 0.1% Triton X-100 (in PBS) containing Hoechst and Alexa-488-conjugated phalloidin where indicated. Coverslips were placed on glass slides and samples were preserved with aqua polymount mounting medium (ID#18606, Polyscience).
Fluorescence microscopy was performed using a 63×/1.4 C-Plan Apo oil immersion lens on a Zeiss LSM 880 confocal microscope driven by ZEN Black software (v2.3). Microscopy images were obtained using z-stack image series taking 8–16 slices.
Confocal images were processed using Airyscan processing (Weiner filter) using “Auto Filter” and “3D Processing” options.
Fluorescence microscopy was performed using a 63×/1.4 C-Plan Apo oil immersion lens on a Zeiss LSM 880 confocal microscope driven by ZEN Black software (v2.3). Microscopy images were obtained using z-stack image series taking 8–16 slices.
Confocal images were processed using Airyscan processing (Weiner filter) using “Auto Filter” and “3D Processing” options.
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