PAM were grown on coverslips and treated or untreated with cGAMP (20 μg/ml) or mock or ASFV infected at the indicated MOI. At the indicated times postinfection (1 or 6 hpi) or 1 h after cGAMP treatment, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.2% Triton X-100 for 15 min, and blocked with PBS–5% BSA for 45 min. The cells were stained with the primary antibodies anti-STING antibody (1/50) and anti-γ-adaptin (AP1) antibody (1/100) diluted in PBS–1% BSA for 1 h. The cells were then washed with PBS and incubated with the fluorescence-conjugated secondary antibodies anti-rabbit/Alexa Fluor 488 (1/500) and anti-mouse/Alexa Fluor 555 (1/500) diluted in PBS–1% BSA for 1 h. After a wash with PBS, the coverslips were mounted with DAPI Fluoromount-G (SouthernBiotech). Images were acquired by using a CLSM LSM710 coupled to an inverted AxioObserver microscope (Zeiss) with a 63× oil immersion objective lens and analyzed using ImageJ. Colocalization between STING and AP1 was quantified by calculating Mander’s coefficient (M1) using the plugin JACoP (Just Another Colocalization Plugin; Fiji).
Inverted axioobserver microscope
Manufactured by Zeiss
Sourced in Germany
The Inverted AxioObserver microscope is a versatile laboratory instrument designed for a range of imaging and analysis applications. It features an inverted optical configuration, allowing for easy access to the sample and accommodating a variety of specimen types. The microscope's core function is to provide high-quality, detailed images of samples under investigation.
Automatically generated - may contain errors
Lab products found in correlation
0.05 μm polystyrene microspheres, by Polysciences (2 mentions)
Micropoint pulsed nitrogen, by Lumencor (2 mentions)
Clsm lsm710, by Zeiss (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Fura 2 am, by Zeiss (1 mentions)
Definite focus, by Zeiss (1 mentions)
Zen blue, by Zeiss (1 mentions)
5 protocols using inverted axioobserver microscope
1
STING-AP1 Colocalization Analysis
2
Exopher Photobleaching Dynamics Analysis
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Synchronized Is[Pmec-4mCh1] adult day 2 animals were immobilized on 7.5% M9 agarose pads with 2.5 μL PolySciences 0.05 μm polystyrene microspheres. Exopher centers were photo-bleached with 7 pulses of the MicroPoint pulsed nitrogen pumped dye laser (neutral density filter at position 9, Lumencor solid state light source) attached to a Zeiss Inverted Axio Observer microscope (100x 1.4 N.A. objective) on an anti-vibration table. 1 frame was recorded every 5 seconds using constant excitation intensity and exposure time with a Qimaging EXi Blue camera. Images were analyzed with ImageJ. Exopher fluorescence intensity was normalized to the intensity of the first data point in each series.
3
Fura-2AM Calcium Imaging in Cortical/Hippocampal Neurons
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Mouse cortical/hippocampal neurons (19–23 DIV) were loaded in neurobasal containing 20 μm Fura-2AM (Invitrogen) for 30 min. After two washes in physiological 1.6 mM calcium-containing buffer (139 mM NaCl, 1.25 mM glucose, 15 mM Na2HPO4, 1.8 mM MgSO4, 1.6 mM CaCl2, 3 mM KCl, 10 mM HEPES), Fura-2AM-loaded neurons were imaged at 37 °C on an inverted AxioObserver microscope (Carl Zeiss) equipped with a 300 W Xenon lamp (Suttler instruments) and a Fluar 40× (numerical aperture (NA) 1.4) oil immersion objective. Fura-2AM was sequentially excited at 340 and 380 nm and the emission monitored at 510 nm. Images were acquired with a cascade 512 EMCCD camera every 2 s and digitized using Metafluor software (Roper scientific). The intracellular calcium concentration was estimated by measuring the F340/380 nm ratio of fluorescence. Neurons were treated for 40 s with 100 μM DHPG in 1.6 mM calcium-containing buffer.
4
Exopher Photobleaching Dynamics Analysis
5
Automated Quantification of SOD1 Aggregation
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Images were acquired using an inverted AxioObserver microscope (Carl Zeiss AG, Germany) with a motorized stage, and exported for analysis as a high resolution JPG files. For these studies, we used the Zen Blue (Carl Zeiss AG, Germany) to prepare a 96 well template with four global focus points. The template was calibrated for every new plate, and each focal point was adjusted in order to determine the focus plane of the plate. Our focusing strategy, along with the Definite Focus (Carl Zeiss AG, Germany), ensured that each well is then in focus. In order to quantify induced aggregation of SOD1 using our reporter protein, we developed an ImageJ-based algorithm that reliably counts inclusions based on the area that inclusions occupy normalized to total expressed GFP (S1 Fig ). Briefly, to count inclusions and their area, the algorithm first converts each image into 8-bit images, performs a local background subtraction, thresholds based on pre-set settings, and counts the number of inclusions (and their size) based on a predefined size. In order to normalize induced aggregation, a parallel algorithm estimates the total area occupied by reporter protein fluorescence with a low threshold. The percentage of aggregation is reported as a ratio of area of inclusions divided by total area of fluorescence.
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