Leica application suite advanced fluorescence software

Third instar larvae were selected, dissected in PBS, fixed in 4% formaldehyde (Ted Pella) in PBS for about 15 minutes and washed ×3 in 0.1% Triton X-100 in PBS. FITC-conjugated anti-HRP (Jackson ImmunoResearch Laboratories) was used at 1:150. For Dlg staining, monoclonal mouse anti-Dlg (1:500)21 (link) and Alexa Fluor 594 nm conjugated goat anti-mouse IgG antibodies (Molecular Probes/Life Technologies) were used. Laval preparations were mounted in SlowFade Antifade kit (Invitrogen). Confocal images were collected from Leica TCS SP5 AOBS confocal microscopes equipped with 40× or 100× inverted NX oil lens, located at Korea Basic Science Institute (Gwangju, Korea). Leica Application Suite Advanced Fluorescence software was used to capture, process and analyze images. Analysis of the NMJ was performed essentially as described21 (link).

Wandering third instar larvae of adult flies were randomly selected, dissected in PBS and then fixed in 4% formaldehyde in PBS for approximately 15 min. After blocking with 5% normal goat serum in PBS-T (0.3% Triton X-100) for 1 h, the antibodies with 5% normal goat serum in PBS-T were incubated with the fixed larvae for approximately 1.5 h at room temperature. Larval preparations were mounted with a SlowFade Antifade Kit (Invitrogen). NMJ images were visualized using a laser scanning confocal microscope system (TCS SP5 AOBS/Tandem microscope, Leica-Microscope Systems GmbH, Germany) at Korea Basic Science Institute, Gwangju Center. Leica Application Suite Advanced Fluorescence software was used to analyse images. We performed the analyses of NMJs essentially as described [38 (link)].

Immediately after isolation, tumors from in vivo models were fixed using 4%-paraformaldehyde followed by 30% sucrose saturation after which tumors were frozen and 20-µm-thick sections were used for stainings.
For in vitro immunofluorescent imaging, cells growing on glass coverslips were fixed using 4%-paraformaldehyde and permeabilized using 1% Triton X100. Tissue sections or fixed cells were incubated with the indicated primary antibody for 3 h followed by incubation with fluorescently labeled secondary antibodies for 1 h, mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific), and imaged using an SP8-X confocal microscope (Leica) and the Leica Application Suite-Advanced Fluorescence software. To detect nuclei, sections were counterstained with Hoechst 33342 (Sigma) (405 nm laser), for F-Actin detection ActinGreen-488 (phalloidin) ready probe (ThermoFisher, 1:1000) (488 nm laser) was used. For HE stainings, frozen tumor sections were stained with hematoxylin and eosin (H&E). The following primary antibodies were used: rabbit anti-MSN (HPA011135, Sigma, 1:100). As secondary antibody goat-anti-rabbit-Alexa546 (A11035, Invitrogen, 1:500) was used.

Confocal microscopy was performed using a Leica TCS-SP8 confocal microscope (Leica Microsystems). mKATE2 was exited at 588 nm and detected at 618 to 653 nm. GFP was excited at 488 nm and detected at 498 to 524 nm. Calcofluor white was excited at 405 nm and detected at 429 to 499 nm. AF488 was excited at 488 nm and detected at 506 to 535 nm. AF594 was excited at 590 nm and detected at 602 to 640 nm. mCherry was excited at 561 nm and detected at 597 to 635 nm. Aniline blue was excited at 405 nm and detected at 630 to 655 nm. The Leica Application Suite Advanced Fluorescence software was used for image processing. If not indicated otherwise, images are horizontal projections of z-stacks.
Epifluorescence microscopy was performed with a Zeiss Axioplan 2 imaging microscope (Carl Zeiss AG) equipped with a CoolSNAP-HQ charge-coupled-device camera (Photometrics) and controlled by the imaging software MetaMorph (Universal Imaging). GFP was observed using GFP filters (ET470/40BP, ET495LP, and ET525/50BP) (Semrock). AF594 was observed using rhodamine filters (HC562/40BP, HC593LP, and HC624/40BP) (Semrock). DAPI staining was detected using the DAPI filter sets (HC375/11BP, HC409BS, and HC447/60BP) (Semrock). Image processing was performed with ImageJ (https://imagej.nih.gov/ij/).

For each of the markers studied, fluorescence microscopy was applied for HDF-a, human skin sections and the respective positive and negative controls. Images from human skin sections have been taken from those areas where some staining was visible. In general, the dermal part of skin sections have been shown, because staining—when present—was in the dermal part of the skin.
Digital images were obtained with an Olympus IX70 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a Leica DFC340 FX camera using Leica Application Suite Advanced Fluorescence software (LAS AF). All images were acquired within simultaneous series and digitally stored using the same settings.

MCF7 cells growing on coverslips were serum starved for 72 hours and then treated for EGF for 8 hours. Cells were washed in PBS and then fixed for one hour at room temperature in 4% paraformaldehyde. After 3 washes in PBS, cells were permeabilized in 0.1% Triton-X 100 for 20 minutes. and washed 3X in PBS. After blocking in 2% BSA, coverslips were incubated with MdmX and/or Mdm2 antibodies (Red Mdm2 – N20, Green Mdm2 – SMP14, Red MdmX – Bethyl, Green MdmX – 8C6) overnight at 4° C and washed 3X in PBS. Coverslips were then incubated with AlexaFluor 488 and 637 linked to anti-mouse and anti-rabbit secondary antibodies (respectively) for 1 hour, washed 3X in PBS, mounted on slides with Prolong Diamond Antifade Mountant with DAPI and visualized with a Leica SP8 MP confocal microscope at room temperature. Images were obtained using HC PL APO 40x/1.3 oil CS2 objective and the Leica Application Suite Advanced Fluorescence Software provided by the Indiana Center for Biological Microscopy core.