Primary rat cortical/hippocampal neurons were plated on coated 2 cm2 glass coverslips following the manufacturer's protocol (BrainBits®). All drug applications were performed blindly. After 7 days of differentiation, neurons were treated with the following concentrations: ADDL (1 μM), CAT-SKL (1 μM), Wy-14,643 (100 μM). Slides were washed three times with Hank's balanced salt solution between treatments, and then fixed and stained with the appropriate antibodies. Primary and secondary antibodies employed include: anti-MAP-2 (1:800) (Millipore) coupled with AlexaFluor 488 goat anti-mouse (1:500) (Life Technologies); and anti-PMP70 (1:500) (abcam®) coupled with AlexaFluor 546 goat anti-rabbit (1:15000) (Invitrogen). Microscopic analysis was performed using the Leica TCS SP2 confocal microscope located in the Department of Anatomy and Cell Biology's microscope facility at the Wayne State University School of Medicine, and Zeiss ApoTome Imaging System from MICR, also at the Wayne State University.
Apotome imaging system
Manufactured by Zeiss
Sourced in Germany
The ApoTome Imaging System is a microscopy tool designed to capture high-quality, optical sectioning images. It uses a structured illumination technique to improve the contrast and resolution of fluorescence microscopy images, enabling the visualization of fine subcellular structures.
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Lab products found in correlation
Axio observer microscope, by Zeiss (10 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Fluorescent mounting medium, by Agilent Technologies (4 mentions)
Axiovert 200m, by Zeiss (3 mentions)
Axiovision software, by Zeiss (3 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (2 mentions)
Poly d lysine, by Merck Group (2 mentions)
Alexa fluor 647 anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 anti rabbit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
T20922, by Thermo Fisher Scientific (2 mentions)
Axiocam mrm camera, by Zeiss (2 mentions)
Tcs sp2 confocal microscope, by Leica (1 mentions)
Alexa fluor 546 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Anti map2, by Merck Group (1 mentions)
Anti pmp70, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine kit, by Thermo Fisher Scientific (1 mentions)
Zen blue software, by Zeiss (1 mentions)
Axiocam mrm, by Zeiss (1 mentions)
28 protocols using apotome imaging system
1
Assessing Neuronal Morphology under Stress Conditions
2
Plasmid DNA and Morpholinos Microinjection
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RNA for microinjection was prepared using the mMessage mMachine Kit (Life Technologies, CA, USA). Injection amounts were 200 pg for ca camkII (camkII T286D), dn camkII (camkII K42M), and for myc-arrb2, myc-arrb1, HA-gβ1 (HA-gnb1), and HA-gγ2 (HA-gng2); injection amounts were 500 pg for pkcα, dn pkcα, β-ARKct, and 1 ng for pkcα-gfp and fzd7. For ca rhoA 5 pg and ca rac1 10 pg plasmid DNA were injected. Knock-down was achieved by injection of the following antisense Morpholino oligonucleotides: Dvl2 MO, Dvl1 MO, and Dvl3 MO (0.8 pmol each), Arrb2 MO1 (0.8 pmol), Arrb2 MO2 (0.8 pmol), Fzd7 MO (4 pmol). Pertussis Toxin (PTX) was added to the culture medium at 100 ng/ml.
Embryos were injected at the two-cell stage for Animal Cap explants or at the four-cell stage in both dorsal blastomeres for Keller explants and cultured until they reached stage 10 or 10.5, respectively.
Keller open face explants were prepared and cultured as described in [18] (link). Explants were scored as “fully elongated” if they showed >75% elongation, as “partially elongated” if elongation was between 25% and 75% and “not elongated” if explants showed less than 25% elongation when compared to fully elongated control explants. Immunofluorescence staining of Animal Cap explants was performed as described previously [21] (link). Photographs were taken on a Zeiss Apotome imaging system (Zeiss, Oberkochen, Germany).
Embryos were injected at the two-cell stage for Animal Cap explants or at the four-cell stage in both dorsal blastomeres for Keller explants and cultured until they reached stage 10 or 10.5, respectively.
Keller open face explants were prepared and cultured as described in [18] (link). Explants were scored as “fully elongated” if they showed >75% elongation, as “partially elongated” if elongation was between 25% and 75% and “not elongated” if explants showed less than 25% elongation when compared to fully elongated control explants. Immunofluorescence staining of Animal Cap explants was performed as described previously [21] (link). Photographs were taken on a Zeiss Apotome imaging system (Zeiss, Oberkochen, Germany).
3
Quantitative Analysis of Placental JAM-C
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Zeiss Apotome Imaging System (Carl Zeiss, Germany) and Zen blue software (Carl Zeiss, Germany) were used to examine and capture placental tissue respectively at an initial objective magnification of × 40 within four fields of view. Quantification of JAM-C expression was performed for each field of view. The placental villi field of view was opened in Image J software [Fiji version, National Institute of Health (NIH), Wisconsin, USA] and underwent color deconvolution into the green channel (Fig. 1C), red channel (Fig. 1D), and blue channel (Fig. 1D). The threshold of chromogen (brown stain) in the red channel was calculated as a percentage expression of JAM-C in the frame, and the threshold expression of hematoxylin (blue stain) in the blue channel was calculated as a percentage expression of villi area in the frame (Fig. 1 F-G). The percentage expression of JAM-C per villi area in the frame was calculated as [%brown/(%brown + %blue) × 100]. The immuno-reactivity was expressed as a percentage of JAM-C label within the conducting villi and exchange villi, respectively.
4
Immunostaining and Imaging of Chicken Pox Virus Antigens
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1ml of CPKs were fixed with 1% paraformaldehyde at 4°C for 24 hrs and rinsed with PBS. CPKs were plated by centrifugation at low speed (500 × g; 800rpm) for 5 min on 0.17μm-thick- glass coverslips using a Cytospin 4 devise (ThermoFisher Scientific). For MDV antigens, the immunostainings were performed in a blocking solution (PBS, 0.1% Triton X-100, 1% bovine serum albumin) for 1 h at room temperature, then incubated for 45 min with a GAM secondary antibody coupled to Alexa Fluor 594 and finally nuclei counterstaining with Hoechst 33342 dye (1:2000e) (Invitrogen) and mounting with Mowiol (Merck). Samples were assessed using an Axiovert 200 M inverted epifluorescence microscope equipped with an Apotome imaging system (Zeiss). Images were captured with an AxioCam MRm charge-coupled-device camera (Zeiss) using the Axiovision software (Zeiss).
5
Immunocytochemistry of Cell Lines
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Glass cover slips were coated with PDL (Sigma-Aldrich) for HeLa cells and fibroblasts while growth-factor reduced matrigel (Millipore, 1/1,000 in PBS) was used for iNeuron experiments. Cells were fixed with 4% (w/v) paraformaldehyde (PFA), permeabilized with 1% Triton-X-100, blocked with 10% goat serum and incubated with primary antibodies followed by Alexa-488, -568 or -647 conjugated secondary antibodies (1/1,000, Invitrogen). Nuclei were stained with Hoechst 33342 (1/5,000, Invitrogen). Tyramide signal amplification (T20922, Invitrogen) was used for PINK1 staining. Coverslips were mounted onto microscope slides using fluorescent mounting medium (Dako). High-resolution confocal fluorescent images were taken with an AxioObserver microscope equipped with an ApoTome Imaging System (Zeiss).
6
Fluorescence Microscopy of GPR1 Signaling
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For fluorescence microscopy, HEK293 cells were seeded into 8 well 15µ-slides (Ibidi, 140,000 cells/200 µL/well) coated with poly D-lysine and grown overnight. Next, cells were transfected with 900 ng of the respective GPR1-eYFP plasmid and, where applicable, 100 ng of either rab4-CFP, rab11-CFP, or mCherry-arrestin3. Transfection was achieved using Lipofectamine 2000 according to the manufacturer’s protocol. One day post-transfection, fluorescence microscopy experiments were performed on an AxioVision Observer.Z1 microscope equipped with an ApoTome imaging system (Zeiss, Jena, Germany). Before the experiment, cells were starved in OptiMEM reduced serum medium containing Hoechst 33342 for 30 min. To observe arrestin recruitment, cells were stimulated with 1 µM chemerin-9 in OptiMEM for the indicated period. To observe peptide uptake, cells were stimulated with 1 µM Tam-chemerin-9 in OptiMEM, which was replaced with acidic wash (50 mM glycine, 100 mM NaCl, pH 3) in HBSS after the indicated time, followed by two washing steps with OptiMEM. Microscopy was carried out in OptiMEM; the exposure time was held constant whenever changes over time were observed.
7
Quantitative Imaging Analysis of Neuronal Markers
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Brightfield images were captured using the Aperio slide scanner (Vista, CA, USA). Fluorescent images were taken with a 40 x Plan-Apochromat objective using a Zeiss AxioObserver equipped with an ApoTome Imaging System (Zeiss). Microglial and astroglial cell counts and morpholological analysis (process length and cell body size) were quantified using MetaMorph Image Analysis Software (Molecular Devices) with the neurite outgrowth application module [4 ]. MetaMorph Software with the cell counting module was used to assess the burden of NeuN positive neurons. First, ImageScope® software (v12.1; Leica Biosystems) was used to annotate the cortex on mid-sagittal sections stained for NeuN for each mouse. Then, Positive Pixel Count Algorithm was established to recognize and quantify NeuN positive cells .The output parameter was the number of NeuN-positive neurons per given mm2 area annotated.
8
Immunofluorescence Staining of Catalase
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Neurons were plated onto poly-D-lysine coated coverslips as described above and fixed for 10 minutes in 3.7% paraformaldehyde and permeablized with 0.1% Triton X-100. Sites that non-specifically bind to antibodies were blocked with 5% normal goat serum for 15 minutes. Cells were then incubated with rabbit anti-catalase antibodies (1:100) for 1 hour, washed with 0.01% Tween-20 in phosphate buffered saline, and incubated with goat anti-rabbit Alexafluor 546 antibodies (1:15000) for 1 hour. Neurons were then mounted with ProLong®Gold antifade reagent with 4′,6-diamidino-2-phenylindole (Invitrogen). Microscopic analysis was performed using the Zeiss ApoTome Imaging system from the Microscopy, Imaging and Cytometry Resources Core Facility (MICR), at the Wayne State University School of Medicine.
9
Immunofluorescence Microscopy of Mitochondria and Autophagy Markers
10
Membrane Localization of Mutant GHSR Variants
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