At indicated time points, infected cells were fixed for 20 min at room temperature with 4% (w/v) paraformaldehyde (in PBS). After washing in PBS, samples were first blocked in PBS containing 5% (v/v) FBS and 0.05% (w/v) saponin (Sigma-Aldrich) (blocking buffer). Samples were then stained using primary antibodies diluted in blocking buffer: 1:50 anti-LAMP-1 clone H4A3, supernatant (H4A3 was deposited to the DSHB by August, J.T. / Hildreth, J.E.K. (DSHB Hybridoma Product H4A3)) and 1:10,000 rabbit anti-C. burnetii antibodies (Roy Laboratory, Yale University). Secondary antibodies, anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific) were used at a dilution of 1:3,000 in blocking buffer. DNA was stained using Hoechst 33258 (1:10,000; Thermo Fisher Scientific) and coverslips were mounted onto glass slides using ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Fluorescence microscopy was performed using a Nikon A1Si confocal microscope, and images were acquired using NIS-Elements software (Nikon).
A1si confocal microscope
Manufactured by Nikon
Sourced in Japan
The A1Si confocal microscope is a high-performance imaging system designed for advanced biological and materials science research. The core function of this microscope is to provide high-resolution, three-dimensional imaging of samples by using a focused laser beam to scan across the specimen, capturing images at multiple focal planes. The A1Si features a sensitive detection system and offers superior image quality, making it a versatile tool for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Goat serum, by Thermo Fisher Scientific (4 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (3 mentions)
Nis elements ar imaging software 4, by Nikon (2 mentions)
Matrigel, by Corning (2 mentions)
Nis elements c software, by Nikon (2 mentions)
Ab5076, by Abcam (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Anti satb1, by Abcam (1 mentions)
Goat anti rabbit igg tritc, by Jackson ImmunoResearch (1 mentions)
Prolong gold antifade mountant, by Nikon (1 mentions)
Ab33451, by Abcam (1 mentions)
Af3628, by R&D Systems (1 mentions)
Sc 515777, by Santa Cruz Biotechnology (1 mentions)
Gb13044, by Wuhan Servicebio Technology (1 mentions)
41 protocols using a1si confocal microscope
1
Immunostaining of Coxiella-Infected Cells
2
Visualization of PTH Receptor Trafficking
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Stable transfected HEK293 cells were cultured on poly-l -lysine–coated glass coverslips for 48 h and serum starved for 1 h before stimulation with PTH(1-34) (100 nM) or PTH−TMR (100 nM) over a time course of 0–120 min. After 15 min of stimulation, cells were washed twice with 1× PBS and replaced with complete medium. Cell surface PTH−TMR fluorescence was detected by subjecting cells to an ice block (4°C for 10 min) and washing and then fixing them (4% paraformaldehyde [PFA]). Cells were then permeabilized with 0.1% Triton X-100 and/or 0.1% saponin and stained with indicated antibodies. Samples were mounted in ProLong Gold Antifade (Thermo Fisher Scientific) and imaged using a Nikon A1Si confocal microscope equipped with a 10× (dry) lens and a 60× (oil) lens (Nikon, Tokyo, Japan). Time-lapse confocal microscopy was performed under controlled atmospheric conditions (37°C and 5% CO2/air) in a Tokai Hit (Fujinomiya, Japan) Stage Top incubator (INUG2E-TIZ) as previously described (Ng et al., 2013 (link)).
3
Apoptosis and Necrosis Detection in Stroke Model
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PI/TUNEL staining was performed as previously reported52 (link). Briefly, PI was intraperitoneally injected (1 μg/g; Sigma-Aldrich, St. Louis, MO, USA) while the tMCAO model was established. Cell apoptosis was detected via TUNEL (Roche, Mannheim, Germany) staining. Frozen brain sections were incubated with TUNEL (37 °C for 1 h), washed three times with PBST (PBS with 0.5 ‰ Tween-20), and then mounted and visualized with a Nikon A1-Si confocal microscope (Nikon, Tokyo, Japan). As reported previously, PI−/TUNEL+ and PI+/TUNEL+ indicate apoptosis and necrosis, respectively53 (link).
4
SATB1 Localization in Renal Cell Carcinoma
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Immunofluorescence staining with the antibody to SATB1 was performed to determine the expression and distribution of SATB1 in human RCC cell lines. Cells cultured on a 24-well plate were fixed in 4% paraformaldehyde for 20 min at 37°C, and then washed twice with PBS for 10 minutes. After permeabilized with 0.3% Triton for 15 minutes, cells were blocked with 5% BSA for 30 min. Subsequently, cells were incubated with a rabbit polyclonal anti-SATB1 (1∶200; Abcam Inc., Cambridge, MA, USA) overnight at 4°C, followed by visualization with a goat anti-rabbit IgG-TRITC (1∶100; Jackson, West Grove, PA, USA) for 1 hour at room temperature in the dark. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min. Images were evaluated with a Nikon-A1Si confocal microscope (Nikon Corporation, Tokyo, Japan).
5
Quantifying Osteoclast Formation and Actin Organization
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BMMs were seeded in a 24-well plate with coverslips (13-mm) and induced by 50 ng/ml hRANKL with or without i-bodies, until the emergence of multinucleated osteoclasts. Cells were fixed in 4% w/v paraformaldehyde for 10 min at RT and permeabilized by 0.1% v/v Triton X-100 for 5 min before 1-h incubation with 3% w/v BSA PBS for blocking. After washing with 0.2% w/v BSA-PBS three times, cells were incubated with rhodamine–phalloidin (1:300 diluted in 0.2% w/v BSA-PBS) for 2 h at RT. Cells were then gently washed four times in 0.2% w/v BSA-PBS and PBS in sequence before staining with 4′,6-diamidino-2-phenylindole (1:10,000 diluted in PBS) for 5 min. After washing with PBS, stained cells were visualized via a Nikon A1Si Confocal Microscope (Nikon Corporation).
6
Histological Analysis of Liver Samples
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The sections of formalin-fixed, paraffin-embedded liver samples were stained with hematoxylin and eosin for standard histology. For the assessment of collagen deposition, Sirius Red staining was performed using the staining assay kit according to the manufacturer’s instructions. For immunohistochemical staining or immunofluorescent staining, the slides were incubated with primary antibodies (Table S1 ) followed by appropriate secondary antibodies (Table S1 ). For immunofluorescent staining,the slides were mounted with DAPI-containing medium and the images were acquired with a Nikon-A1-si confocal microscope.
7
Osteoclast Formation and Cytoskeletal Analysis
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5 × 104 BMMs were seeded onto glass coverslips (14 mm in diameter) in 24‐well plates per well. The BMMs were then induced to form mature osteoclasts, as described previously, in the presence or absence of D.P (30 μmol/L). The cells were then fixed and permeabilized with 0.1% (v/v) Triton X‐100. After blocking unspecific binding using 3% BSA in PBS, the cells were incubated with anti‐vinculin antibody (1:500) or anti‐NFATc1 antibody (1:500) at 4°C overnight. A secondary antimouse IgG conjugated with FITC was used to produce a fluorescent signal. After the incubation of secondary antibody for 1 hour, the cells were stained with rhodamine phalloidin for 20 minutes. Last, the cells were incubated with DAPI or Hoechst 33258 dye for 10 minutes and mounted to glass slides using the ProLong Gold Antifade Mountant and visualized using NIKON A1Si confocal microscope (Nikon Corporation).
8
Immunofluorescent Staining of Immune and Vascular Markers
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Immunofluorescent staining was performed as previously described [65 (link)]. The primary antibodies used were as follows: cGAS (Mouse, 1:100, sc-515777, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MOMA-2 (Rat, 1:100, ab33451, Abcam, Cambridge, UK), CD31 (1:100, AF3628, R&D Systems, Minneapolis, MN, USA) and α-SMA (Rabbit, 1:100, GB13044, Servicebio, Wuhan, China). 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA) was applied to stain nuclei. Fluorescent images were captured with a Nikon A1Si confocal microscope (Nikon, Tokyo, Japan), and analyzed with NIS Elements AR Imaging Software 4.10 (Nikon) and ImageJ 1.41 software (NIH, Bethesda, MD, USA).
9
Matrigel Plug Assay for Angiogenesis
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All experiments with mice were performed under protocols approved by the institutional animal care and use committee of Shanghai Jiao Tong University. Matrigel plug assay was performed as described previously (Chen et al. 2017 (link)) using 6- to 8-wk-old male nude mice (Si Lai Ke Experimental LLC). In brief, 1 × 106 HUVEC cells at passages 4–6 and 2 × 106 human mesenchymal stem cells (Lonza) were mixed with 200 µL ice-cold Matrigel (Corning 356237) and injected subcutaneously. After a week, the Matrigel plugs were dissected out, fixed with 4% paraformaldehyde overnight, and paraffin embedded. HUVECs were stained using Ulex europaeus agglutinin I (1:200, Vector Labs), a lectin that recognizes human but not mouse endothelial cells. Blood vessels from host mice were stained with PECAM1 (1:200, BD Biosciences). Proliferating cells were stained using Ki-67 antibody (1:200, Abcam), and apoptotic cells were stained using antiactivated caspase 3 antibody (1:200, Millipore). Images were acquired with a Nikon A1Si confocal microscope. The percentage of proliferating or apoptotic endothelial cells and the total vascular area were measured using ImageJ.
10
Lipid Accumulation in Macrophages: Oxldl and RU.521
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RAW264.7 macrophages were incubated in DMEM containing oxLDL (100 μg/mL, YB-002, Yiyuan Corporation) with or without RU.521 (2 μg/mL) for 24 h. After fixation for 20 min, cells were blocked with 1% BSA, followed by staining with BODIPY 493/503 (D3922, Invitrogen) for 30 min. Next, nuclei visualized by staining DAPI for 10 min. Finally, antifade mounting medium (P0126, Beyotime Institute of Biotechnology, Jiangsu, China) was applied to prevent fluorescence quenching. Images were acquired using a Nikon A1Si confocal microscope. Analysis of different images was performed using ImageJ and Adobe Photoshop CC. Oil Red O staining was carried out as previously described [66 (link), 67 (link)]. RAW264.7 macrophages were incubated in DMEM containing oxLDL (100 μg/mL) with or without RU.521 (2 μg/mL) for 24 h. After fixation for 30 min, cells were stained with oil red O (G1015, Servicebio) for 30 min, and then visualized by light microscopy (Olympus, Tokyo, Japan).
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