To visualize KCC2 immunofluorescence and tdTomato or eGFP, Z-stack images were obtained with a Zeiss LSM 700 confocal module configured to an Axio Observer Z.1 with a 20x (0.8 NA) or 63x (1.40 NA) objective (Zeiss, Oberkochen, Germany) and Zen 2010 software (Carl Zeiss INC, Thornwood, NY). 3-D reconstruction and colocalization of KCC2 and tdTomato- (D1R-expressing MSNs) or eGFP- (D2R-expressing MSNs) containing voxels from 20x objective Z-stacks (0.31 µm × 0.31 µm × 0.79 µm) was performed with Imaris software (Bitplane, South Windsor, CT). Colocalization thresholds were determined using secondary control (without primary antibody incubation) sections from the same mice. Thresholds were equivalent across all mice and set to < 1% colocalization between tdTomato/eGFP and KCC2 immunofluorescence for control slices. Colocalization results are displayed as the percentage of voxels with tdTomato or eGFP fluorescence colocalized with voxels containing KCC2 immunofluorescence. High magnification (63x objective) images were obtained to visualize KCC2 subcellular localization.
Lsm 700 confocal module
Manufactured by Zeiss
Sourced in Germany
The LSM 700 confocal module is a precision optical instrument designed for high-resolution imaging and analysis of microscopic samples. It utilizes a focused laser beam to scan the sample and generate detailed, three-dimensional images of the specimen. The LSM 700 incorporates advanced optics and detection systems to provide a powerful tool for researchers and scientists conducting in-depth studies of biological, materials, and other micro-scale phenomena.
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Lab products found in correlation
Axio observer, by Zeiss (5 mentions)
Axio observer z1, by Zeiss (3 mentions)
Axio observer inverted microscope, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Zen 2010, by Zeiss (1 mentions)
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
35 mm petri dish, by Greiner (1 mentions)
Prolon gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Axioplan 2 immunofluorescence microscope, by Zeiss (1 mentions)
Dmem 4.5 g l glucose, by Lonza (1 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Fbs superior, by Harvard Bioscience (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Cover glasses, by Marienfeld (1 mentions)
L glutamine, by Lonza (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
780 quasar confocal module, by Zeiss (1 mentions)
13 protocols using lsm 700 confocal module
1
Visualizing KCC2 Distribution in MSNs
2
Visualizing Adherens Junctions in MDCK Cells
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MDCK RFP-E-Cadherin cells were seeded at confluency on a 2 mg/mL collagen matrix in a 35 mm Petri dish (Greiner bio-one, Kremsmünster, Germany). Cells were transfected with p60AmotL2-GFP, p60AmotL2ΔILI-GFP plasmid, or GFP-myristylated for 8h or more before obtaining confocal images. NucBlue™ Live ReadyProbes™ Reagent (product number R37605, Thermo Fisher Scientific, Waltham, MA, USA) was used to visualize nuclei. Images were captured using a Zeiss AxioObserver with LSM700 confocal module in 40× or 63× objective (Zeiss, Oberkochen, Germany). During the experiments, Petri dishes were maintained in a confocal integrated incubator (37 °C, 5% CO2, and a humid atmosphere). Images were captured every 30 min for at least 12 h. Image and video captures were processed and analyzed using the IMARIS software (https://imaris.oxinst.com (accessed on 14 January 2019)) and ImageJ software (version 1.53t).
3
Fluorescent Tumor Imaging Protocol
4
Immunofluorescence Analysis of p53, CD44, and iASPP
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hTERT-BJ fibroblasts were fixed in 3% paraformaldehyde for 20 min at room temperature. Thereafter, cells were permeabilized with 0.25% Triton X-100 for 10 min, washed in PBS, blocked by 3% BSA and 5% goat serum in PBS for 1 h, incubated with primary antibodies for 90 min at room temperature, washed four times in PBS, and incubated with secondary antibodies for 1 h. The following primary and secondary antibodies were used at the indicated dilutions and concentration: anti-p53 (1:200; mouse IgG DO-1), anti-CD44 (4 μg/mL; Hermes1), iASPP antiserum (1:200), Alexa Fluor 488 goat anti-mouse (1:1000), and Alexa Fluor 594 goat anti-rabbit (1:1000). Slides were mounted with ProLon gold antifade reagent (Invitrogen), and photographs were taken with a Zeiss Axioplan 2 immunofluorescence microscope and/or a Zeiss LSM 700 confocal module.
5
Bmpr1b Expression in NIH/3T3 Cells
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NIH/3 T3 cells were seeded into a 24-well plate in growth medium on cover glasses (Marienfeld). 24 h later Bmpr1b expression vectors were transfected into the NIH/3 T3 cells using Lipofectamine 2000 (Invitrogen, Life Technologies) following the manufacturer´s instructions. Another 24 h later cells were incubated under serum free conditions (DMEM 4.5 g/l glucose (Lonza), 2 mM L-glutamine (Lonza)) for 1 h, subsequently fixed with 4 % paraformaldehyde in PBS and blocked in PBS containing 10 % FBS superior (Biochrom) over night. Non-permeated cells were incubated with a rabbit anti-HA antibody (H6908, Sigma-Aldrich; diluted 1:100 in PBS containing 10 % FBS superior (Biochrom)) for 1 h. After washing with PBS the cells were incubated with the secondary antibody anti-rabbit-Alexa Fluor 488 (A11008, Molecular Probes Life Technologies) and with DAPI (Invitrogen, Life Technologies) for 1 h. The cover glasses containing stained cells were mounted on microscope slides (‘SuperFrost Plus’, Menzel) using Fluoromount-G (Southern Biotech). Confocal microscopy was performed using Zeiss Axio Imager.M2 equipped with a LSM700 confocal module (Carl Zeiss, 63-fold magnification).
6
MDCK Cell Immunofluorescence Imaging
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MDCK cells were plated at a concentration of 500,000 cells per cm2 and grown to confluency for 4 days in 8-well chamber slides. The slides were then fixed in 4% PFA (product number sc-281692, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and subjected to immunofluorescence staining as described above in the cell immunostaining method. Slides were imaged using a Zeiss AxioObserver with LSM700 confocal module and a 63× objective (Zeiss, Oberkochen, Germany).
7
Multi-Modal Confocal Microscopy Protocols
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Confocal microscopy was performed on a Zeiss AxioObserver with 780-Quasar confocal module & FCS (NIH Grant S10 OD016374), a Zeiss AxioObserver with LSM700 confocal module (NIH Grant S10 OD016374), or a Zeiss Axiovert 200 with 510-Meta confocal module. All DIC microscopy was performed with a Zeiss Axiophot 2 with Janoptik ProgResC14 Plus opitical imaging system.
8
ASC Speck Formation in HCE-2 Cells
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HCE-2 cells were cultured on eight-well chamber slides (Ibidi GmbH, Martinsried, Germany) at a density of 4.5 × 104 cells/well. Sub-confluent (70%-80%) cell layers were transfected with DsRed-ASC plasmid construct (20 ng) along with ExGen500 transfection reagent (Thermo Fischer Scientific, Waltham, MA, USA). The cells were incubated for 24 hours in a humidified 5% CO2 incubator at 37°C followed by a priming with TNF-α (10 ng/mL) for the next 24 hours. Thereafter, ASC specks were observed under the confocal microscope (Zeiss Axio Observer inverted microscope with Zeiss LSM 700 confocal module) at 5 and 24 hours upon UV-B exposure (0.2 J/cm2).
9
Immunofluorescence Staining of FoxP3 and GrB
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Antigen retrieval was performed on deparaffinized sections in 10 mM citrate buffer (pH 6.0) in a pressure cooker for 15 min, followed by a wash in 0.1 M phosphate buffer (PB; pH 7.0). Subsequently, to quench any autofluorescence, the sections were treated with 50 mM glycine for 40 min at room temperature. Thereafter, non-specific staining was blocked with 1% bovine serum albumin for 30 min, followed by an overnight incubation at 4 °C with the primary antibodies against FoxP3 (Abcam, Cambridge, UK) and GrB (both antibodies at a 1:90 dilutions). Next, the sections were washed and incubated for 1 h with the secondary antibodies (1:300, Alexa Fluor 594 anti-rabbit IgG, Cell Signaling and 1:300 Alexa Fluor 488 anti-mouse IgG, Cell Signaling). Nuclei were counterstained with DAPI (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA). Finally, the sections were mounted in Vectashield (Vector H-1000, Vector). The samples were imaged with a Zeiss Axio Observer inverted microscope (×20 or ×40 NA 1.3 oil objectives) equipped with a Zeiss LSM 700 confocal module (Carl Zeiss Microimaging GmbH, Jena, Germany).
10
Confocal Microscopy Imaging Protocol
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Fluorescent images were taken with 40 × (NA 1.3) and 63 × (NA 1.4) oil objectives on a Zeiss Axio Observer equipped with a Zeiss LSM 700 confocal module (Carl Zeiss Microimaging GmBH, Jena, Germany) and a Zeiss XL-LSM S1 incubator with temperature and CO2 control. ZEN software (Carl Zeiss Microimaging GmBH) was used for image processing and 3D rendering.
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