HEK293T cells were co-transfected with full-length FLAG-TREM2 and DAP12 for 24 hr before replating on glass slides. Cells were washed 2x with PBS, then fixed with 4% paraformaldehyde (PFA) in PBS for 5 min. Cells were then washed and blocked with animal-free blocker (Vector Laboratories. Burlingame, CA) for 1 hr at RT. For permeabilized cells, 0.1% triton x-100 was added to the blocking buffer and subsequent steps. Anti-FLAG antibody (M2, Sigma) was added at 1:1000 overnight at 4°C. Cells were then washed twice, and anti-mouse secondary Alexa Fluor 555 conjugate (Life Technologies) was added at 1:200 for 1 hr. Cells were washed a final time and then mounted in VECTASHIELD H-1200 Mounting Medium with DAPI (Vector Laboratories). Confocal microscopy was carried out using a Zeiss LSM 510 META Confocal Laser Scanning Microscope (Carl Zeiss Microscopy, Thornwood, NY) at 400x magnification. The images were acquired with LSM 4.2 software.
Vectashield mounting medium with dapi h 1200
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
Vectashield mounting medium with DAPI (H-1200) is a ready-to-use aqueous mounting medium designed for fluorescence microscopy. It contains the DNA-binding dye DAPI (4',6-diamidino-2-phenylindole) which emits blue fluorescence when bound to DNA, allowing for the visualization of cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Lsm 510 meta confocal laser scanning microscope, by Zeiss (2 mentions)
Ab7800, by Abcam (2 mentions)
A1 confocal microscope, by Nikon (2 mentions)
Lsm software version 2, by Zeiss (2 mentions)
Tmr red, by Roche (2 mentions)
Alexa fluor488 goat anti mouse igg a 11001, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 a 11008, by Thermo Fisher Scientific (2 mentions)
594 a 11012 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Bz 9000 microscope, by Keyence (2 mentions)
Ab150064, by Abcam (2 mentions)
Fv10i, by Olympus (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Animal free blocker, by Vector Laboratories (1 mentions)
Ab96879, by Abcam (1 mentions)
Ab96885, by Abcam (1 mentions)
Ab9026, by Abcam (1 mentions)
Dfc340 fx camera, by Leica (1 mentions)
42 protocols using vectashield mounting medium with dapi h 1200
1
FLAG-TREM2 and DAP12 co-transfection in HEK293T
2
Immunohistochemical Analysis of Epidermal Markers
3
Immunofluorescence Staining of CLCA1 and TMEM16A
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For staining experiments, cells were either transfected or exposed to conditioned medium as described above. Following 24 hr incubation, cells were fixed on glass slides with 4% paraformaldehyde (PFA) in PBS for 5 min and washed twice with PBS. Cells were blocked for 1 hr at room temperature with 1% blocking solution in PBS (Life Technologies) and then incubated with primary antibodies (rabbit anti-human CLCA1 polyclonal antibody 1228 at 1:100 dilution and goat-anti-human-TMEM16A polyclonal antibody S-20 at 1:50 dilution) overnight at 4°C. Slides were washed and incubated with WGA-Alexa Flour 633 conjugate (5 μg/ml) for 30 min at room temperature, followed by secondary antibodies (donkey anti-rabbit IgG-Alexa Fluor 594 conjugate at 1:250 dilution and donkey anti-goat IgG-Alexa Fluor 488 conjugate at 1:200 dilution) for 2 hr at room temperature. Washed slides were then mounted in VECTASHIELD H-1200 Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). Confocal microscopy was carried out using a Zeiss LSM 510 META Confocal Laser Scanning Microscope (Carl Zeiss Microscopy, Thornwood, NY). The images were acquired with LSM 4.2 software and batch processed with AxioVision 4.8.2 (Carl Zeiss Microscopy).
4
Immunostaining of Phosphorylated Signaling Proteins
5
Characterization of FoxP3+ Cells in EC
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In order to characterize the FoxP3-expressing cells, double immunofluorescence staining was performed. 5 specimens of EC-patients were incubated with a mouse anti-FoxP3 antibody (Dilution 1:50; 236AIE7, Thermo Fisher Scientific, Waltham, MA, USA) and a rabbit anti-CD3 antibody (ready to use; N1580, DAKO, Agilent technologies, Santa Clara, CA, USA) or a rabbit anti-CCR4 antibody (Dilution 1:50; HPA031613, Atlas Antibodies, Bromma, SWE) after blocking with Ultra-Vision-Proteinblock (Thermo Fisher Scientific, Waltham, MA, USA). Goat-anti-mouse-Alexa-Fluor488- and Goat-anti-rabbit-Cy-3-conjugated antibodies (both Dianova, Hamburg, Germany) were used as secondary antibodies. Samples were fixed with Vectashield® H1200 mounting medium with DAPI (VectorLab, Burlingame, CA, USA) and analysed using an Axiophot fluorescent photomicroscope (Zeiss, Oberkochen, Germany) and AxioVision 4.8.1 Software.
6
Histological Evaluation of Epidermal Proteins
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EVPOMEs were fixed in 10% neutral formalin. Paraffin-embedded specimens were cut into 5-mm sections and stained with hematoxylin and eosin (HE) for histological examination. For immunohistochemistry, sections were heated in Tris-EDTA buffer (10 mM Tris [Sigma-Aldrich], 1 mM EDTA [Sigma-Aldrich], pH 9.0) for 20 min. After incubating the sections with 5% normal goat serum (Fisher Scientific, Pittsburgh, PA) for 1 h at room temperature, they were incubated overnight at 4°C in a humidified chamber with primary antibodies: mouse monoclonal antibody against filaggrin (ab17808; Abcam, Cambridge, United Kingdom) (1:500), involucrin (ab68; Abcam) (1:5000), p63 (ventana, 790-4509) (no dilution), a rabbit monoclonal antibody against Ki-67 (ab16667; Abcam) (1:200), K15 (ab52816; Abcam) (1:200), and a rabbit polyclonal antibody against GLUT1 (ab14683; Abcam) (1:200). The sections were incubated with the goat anti-mouse IgG (H + L) secondary antibody, DyLight 488 conjugate (Thermo Scientific, Waltham, MA), or goat antirabbit IgG (H + L) secondary antibody, DyLight 594 conjugate (Thermo Scientific), for 90 min in the dark. Glass coverslips were mounted onto slides in Vectashield mounting medium with DAPI (H-1200; Vector Laboratories, Burlingame, CA) for DNA staining.
7
Immunofluorescent Characterization of LAG3 and TLR2 in H4 Cells
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In order to confirm the expression of LAG3 and TLR2 receptors in H4 cells, immunofluorescent (IF) labeling was performed. Cells were cultured on 15‐mm glass coverslips in 24‐well plate without tetracycline for 72 h in accordance with experimental timeline. Next, cells were washed with phosphate‐buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) for 15 min and blocked with 2% normal goat serum for 1 h at room temperature (RT). Cells were incubated overnight at 4°C with primary antibodies (Anti‐Lag3 antibody, Anti‐TLR2 antibody, α‐synuclein antibody) prepared in PBS containing 1% BSA. The next day, cells were washed and treated with Alexa Fluor 488 and 568 secondary antibodies for 1 h at RT. All antibodies used in the study are described in Table 1 . Coverslips were mounted on slides with Vectashield Mounting Medium with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Vectashield Mounting Medium with DAPI H‐1200; Vector Laboratories) and cells were visualized using an Axio observer inverted microscope (Carl Zeiss). Fluorescence intensities of LAG3 and TLR2 receptors were evaluated and intensities were normalized to the total area with ImageJ26 in 13 of the examined areas (Total 13 random areas in three set replicates).
8
Visualization of Internalized Biomolecules
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Following the internalization assay, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min, treated with permeabilization buffer (90% methanol, 5% acetic acid), and then blocked with 10% FBS in PBS for 20 min. The cells were incubated with FITC-labeled streptavidin (ab136201; Abcam, Cambridge, UK) in 10% FBS in PBS for 1 h at room temperature. After washing three times with PBS, the cells were incubated with DAPI for 5 min and mounted using VECTASHIELD Mounting Medium with DAPI (H-1200; Vector Laboratories, Burlingame, CA, USA). Images were acquired using a BZ-X700 fluorescence microscope (KEYENCE, Osaka, Japan). Image processing was performed using Adobe Photoshop CS2.
9
Immunostaining of Drosophila Neuromuscular Junctions
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Immunostaining of body walls was performed as previously described (Ramachandran and Budnik, 2010b (link)). The following primary antibodies were used: 1:500 rat anti-HtsF (Lin et al., 1994 (link); Robinson et al., 1994 (link)) from Dr. Lynn Cooley, 1:5 mouse anti-Hts-1B1 (Zaccai and Lipshitz, 1996 (link)) (1B1 – Developmental Studies Hybridoma Bank), 1:200 rabbit anti-HtsM (Petrella et al., 2007 (link)) from Dr. Cooley, 1:10 mouse anti-HtsRC (Robinson et al., 1994 (link)) (hts RC – DSHB), 1:200 goat anti-Hrp (123-005-021 – Jackson ImmunoResearch), 1:10 mouse anti-Dlg (Parnas et al., 2001 (link)) (4F3 – DSHB), goat anti-phospho-adducin (sc-12614 – Santa Cruz Biotechnology) and 1:500 mouse anti-GFP (G1544 – Sigma-Aldrich). Fluorescent-labeled secondary antibodies from Vector Laboratories were used at a 1:200 dilution. Stained body walls were stored in VECTASHIELD Mounting Medium with DAPI (H-1200 – Vector Laboratories). Images of NMJs at muscles 6/7 from abdominal segment 4 were taken as merged stacks (unless otherwise stated) on a Nikon A1R laser scanning confocal microscope with NIS-Elements software, with experiments and their controls imaged under identical acquisition settings. All images were processed with Adobe Photoshop.
10
Immunofluorescence Staining of Cryostat Sections
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Tissue was embedded in OCT medium (Sakura Finetek, Torrance, CA) and snap frozen in liquid nitrogen. The immunofluorescence staining protocol has been described previously (Cao et al., 2017 (link)). Briefly, the cryostat sections with 8 μm thick were fixed in ice cold acetone for 15 min, rinsing in PBS, 5 min, 3 times, permeabilization in 0.2% Triton X-100 (diluted in PBS) for 5 min. After rinsing in PBS for 5 min, sections were blocked with 3% horse serum for 1 h at room temperature and then incubated with primary antibodies (Table 3 ) at 4°C overnight. The sections were rinsed in PBS for 5 min, 3 times, and then incubated with the secondary antibodies (Table 3 ) for 30 min at room temperature in dark. Sections were finally mounted in VECTASHIELD mounting medium with DAPI (H-1200, VECTOR LABORATORIES, INC. CA, US). The sections were observed under laser scanning confocal microscope (OLYMPUS-FV10i, Olympus Corporation, Tokyo, Japan).
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