For immunofluorescence studies, adipocytes were cultured on 12 × 12 mm poly-L-lysine pretreated slides and then fixed in methanol, followed by rinsing with PBS. The fixed adipocytes were blocked with 1% Bovine serum albumin (BSA) in TBST for an hour and incubated with polyclonal anti-UCP1 antibody (1:500 dilution) overnight at 4°C, followed by three rinses. After that, adipocytes were treated with Fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:500 dilution) in blocking solution. To stain mitochondria, MitoTracker Red (1 mM; Cell Signaling Technology) was used according to the manufacturer's protocol. Then, the cells were fixed, washed once with PBS, and then immunostained. The nuclei of the fixed cells were stained by using Hoechst. Finally, slides were mounted with ProLong Gold Antifade reagent (Life Technologies), and imaging data were obtained by using a Zeiss confocal laser scanning microscope LSM880 (Carl Zeiss, Oberkochen, Germany) combined with Zeiss microscope software ZEN 2012 (Carl Zeiss) and ImageJ software.
Microscope software zen 2012
Manufactured by Zeiss
Sourced in Germany
Zeiss microscope software ZEN 2012 is a platform for controlling and operating Zeiss microscopes. It provides an interface for configuring microscope settings, capturing images, and managing collected data.
Automatically generated - may contain errors
Lab products found in correlation
Lsm880 laser scanning confocal microscope, by Zeiss (1 mentions)
Mitotracker red, by Cell Signaling Technology (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Chemotaxis tool plugin for imagej, by Ibidi (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
2 protocols using microscope software zen 2012
1
Immunofluorescence Staining of Adipocytes
2
Tracking Cell Migration and Mitosis
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Cells were plated on gelatin coated 6 or 12 wells plate and cells were allowed to adhere for at least sixteen hours. Experiments were performed in a humidified chamber with 5% CO2 at 37 ° C in the presence of DMSO or SMIFH2. Cells were imaged every five minutes on a Zeiss Axio Observer Z1 microscope (Carl Zeiss) equipped with a LD Plan-Neofluar Ph2 20x (N.A. 0.40) objective, operated with Zeiss Microscope Software ZEN 2012. Individual cells were tracked using Manual Tracking plugin for ImageJ. Average distance, speed and directionality of movement were computed using the Chemotaxis Tool plugin for ImageJ provided by ibidi GmbH (http://www.ibidi.com ).
Cells entering mitosis were scored manually and defined as follows: a cell entered mitosis when its flat and spread appearance changed to a round-up, yet adhesive state. Initial number of cells was counted manually in each field of observation and percentage of cells entering mitosis every hour was calculated using this initial number of cells as reference.
Cells entering mitosis were scored manually and defined as follows: a cell entered mitosis when its flat and spread appearance changed to a round-up, yet adhesive state. Initial number of cells was counted manually in each field of observation and percentage of cells entering mitosis every hour was calculated using this initial number of cells as reference.
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