Dissection, fixation and immunostaining were performed as described by [68 (link)]. The following antibodies were used: mouse anti-Prospero (MR1A-c, Developmental Studies Hybridoma Bank (DSHB)) at 1:200; mouse anti-Arm (N2 7A1-s, DSHB) at 1:50; rabbit anti-Caspase3 (Cell Signaling, #9661) at 1:300; rabbit anti-DH31 (gift from Jan Veenstra [40 (link)]) and Michael Nitabach [31 (link)]) at 1:500. The secondary antibodies used were anti-mouse Alexa647, anti-rabbit Alexa488, anti-rabbit Alexa546 (Invitrogen). All secondary antibodies were used at 1:1000. Phalloidin-Alexa555 (Molecular Probes, A34055) were used at 1:500 2h at room temperature or 1:2000 overnight at 4°C. Guts were mounted in Fluoroshield-DAPI medium (Sigma) and observed with a Zeiss Axioplan Z1 with Apotome 2 microscope. Pictures in Fig 4M and 4N were acquired using a Zeiss LSM 880 confocal equipped with a Fast AiryScan. Images were analyzed using ZEN (Zeiss) and Photoshop software. Image acquisition was performed at the Microscopy platform of the Institut Sophia Agrobiotech (INRA 1355-UNS-CNRS 7254-Sophia Antipolis).
Axioplan z1
Manufactured by Zeiss
Sourced in Germany
The Axioplan Z1 is a microscope system designed for high-performance imaging and analysis. It features a motorized Z-drive for precise focusing and a modular design that allows for the integration of various accessories and imaging modules. The Axioplan Z1 is a versatile tool for a wide range of applications in research and industrial settings.
Automatically generated - may contain errors
Lab products found in correlation
Apotome 2, by Zeiss (3 mentions)
Fluoroshield dapi medium, by Merck Group (2 mentions)
Efficient navigation zen , by Zeiss (2 mentions)
Alexa555 phalloidin, by Thermo Fisher Scientific (1 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (1 mentions)
Alexa 546, by Thermo Fisher Scientific (1 mentions)
Apotome 2 microscope, by Zeiss (1 mentions)
Lsm 880 confocal, by Zeiss (1 mentions)
Alexa fluor 647 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti ph3, by Merck Group (1 mentions)
Alexa fluor 647 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Bodipy, by Merck Group (1 mentions)
Fluoroshield dapi, by Merck Group (1 mentions)
Vhx 2000, by Keyence (1 mentions)
Axiocam, by Zeiss (1 mentions)
Plan neofluar, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
6 protocols using axioplan z1
1
Comprehensive Immunostaining Procedure for Drosophila Gut
2
Immunostaining of Drosophila Gut Tissue
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Dissection, fixation and immunostaining were performed as described by Micchelli, 2014 (link). Dilutions of the various antibodies were: mouse anti-Armadillo N27A1 at 1:50 (DSHB), mouse anti-Connectin-C1-427 at 1/200 (DSHB), mouse anti-Prospero MR1A at 1:200 (DSHB), rabbit anti-PH3 at 1:1000 (Millipore, 06–570), Rabbit anti-Cleaved Caspase-3 at 1/600 (Cell Signalling Asp175 #9661), Goat anti-mouse AlexaFluor-647 at 1/500 (Molecular Probes Cat# A-21235), Goat anti-mouse AlexaFluor-546 at 1/500 (Molecular Probes Cat# A-11003), Goat anti-rabbit AlexaFluor-647 at 1/500 (Thermo Fisher Scientific Cat# A32733), Goat anti-rabbit AlexaFluor-546 at 1/500 (Thermo Fisher Scientific Cat# A-11010). For microscopy, guts were mounted in Fluoroshield DAPI medium (Sigma, # F6057) and immediately observed on a Zeiss Axioplan Z1 with Apotome 2 microscope. Images were analyzed using ZEN (Zeiss), ImageJ and Photoshop software. Image acquisition was performed at the microscopy platform of the Sophia Agrobiotech Institute (INRAE1355-UCA-CNRS7254 – Sophia Antipolis).
3
Fluorescence Imaging of Gut Microbiome
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R19-S was purchased from Futurechem (FC-8001, Seoul, South Korea). For microscope scanning of the R19-S fluorescence signal, 50 μM of R19-S was added to the 5% sucrose solution (with or without bacteria) deposited on the filter disk. Flies were allowed to feed for 30 min on the mix (5% sucrose + bacteria + R19-S). Then flies were transferred to a new vial with a filter disk only soaked with R19-S in 5% sucrose. Flies were then dissected at the desired time points. The guts were fixed in 4% formaldehyde in PBS for 50 min. Guts were mounted in 80% Glycerol/PBS and immediately observed on a Zeiss Axioplan Z1 with Apotome 2 microscope.
4
Lipid Staining of Larval Guts
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Five days after infection (mid-third instar larvae), ten larval guts were dissected in PBS and fixed for 40 min in 4% formaldehyde in PBS. Guts were then washed twice in PBS and further incubated for 30 min either in 5 µg/mL of BODIPY™ 493/503 (ref D3922, InvitrogenTM Molecular ProbesTM, Waltham, MA, USA) or in a 0.06% Oil-red-O solution (ORO) (ref 189400250, Acros Organics, Fair Lawn, NJ, USA). Guts were then washed twice with PBS. For BODIPY™ labeling guts were mounted in Fluoroshield/DAPI (ref F6057, Sigma) and immediately observed under a fluorescent microscope (Zeiss Axioplan Z1 with Apotome 2). For Oil-red-O labeling, guts were mounted in 80% glycerol/PBS and observed with a numeric microscope (VHX 2000, Keyence).
5
GCaMP6s Fluorescence Imaging in Btk-Intoxicated Midguts
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20XUAS-GCaMP6s/tub-Gal80ts, Dh31-Gal4/+ (GCaMP6s) F1 females were reared at 18°C for 10 days after hatching. Then GCaMP6s females were shifted to 29°C for 5 days before oral intoxication. After 2h of starvation GCaMP6s females were flipped onto fly medium covered with filter disks soaked in sucrose 5% + Btk at 1.108 CFU/5 cm2/fly. Control flies were deposited on a filter disk soaked in 5% sucrose. No bromophenol blue was added to avoid fluorescence attenuation. Flies were left in contact with the filter disks for 30 min, with the exception of the 15 min time point. Midguts were then quickly dissected in PBS 1X and fixed in 4% formaldehyde/PBS for 5 min (not more because a longer time of fixation induces GCaMP6s fading). The guts were rapidly mounted in 50% glycerol/PBS and immediately observed with a Zeiss Axioplan Z1 with Apotome 2. The R2b and the R4c regions were examined for green fluorescence emission.
6
Axioplan Z1 Fluorescence Microscopy Protocol
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Sections were imaged by fluorescence microscopy using a Axioplan Z1 (Zeiss, Germany) epifluorescence microscope. Photomicrographs (1300 × 1030 dpi) were obtained with 2.5× and 5× Plan-Neofluar (Zeiss, Germany) objectives, and acquired using a AxioCam (Zeiss, Germany) digital camera using AxioVision software (v. 4.4; Zeiss, Germany). For colocalization analyses, optical sections were acquired with the Apotome module and a 40× objective. Z-stack photomontage of axonal tracing was done using confocal microscope Zeiss 710. Images were sized using Adobe Photoshop 11 and Illustrator 14.
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