For immunofluorescence microscopy, cells were either fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature or with methanol for 5 min at −20°C. Antibodies were diluted in PBS with 0.1% (v/v) Triton-X100 and 10% (v/v) horse serum. The cells were washed in PBS with 0.1% (v/v) Triton-X100 and incubated for 1 h with primary antibodies. The cells were washed again in PBS with 0.1% (v/v) Triton-X100 and incubated with appropriate secondary antibodies for another 1 h. The cells were washed again first in PBS with 0.1% (v/v) Triton-X100, then PBS, and finally in H2O. In the end, coverslips were mounted onto glass slides using Aqua-poly/Mount mounting medium (Polysciences). Samples were analyzed using widefield fluorescence microscopes (Leica DMRA2 or Zeiss AxioImager D2) or a confocal laser scanning microscope (Zeiss LSM 710 META). Unless otherwise indicated, images were obtained by confocal microscopy and processed using Zeiss LSM Image Browser and Adobe Photoshop software or Fiji (Schindelin et al., 2012 (link)).
Lsm image browser
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The LSM Image Browser is a software tool designed to view and manage digital microscopy images. It provides a straightforward interface for navigating and inspecting image data acquired from laser scanning microscopes.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (12 mentions)
Lsm 510 confocal system, by Adobe (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Axio imager d2, by Zeiss (1 mentions)
Aqua poly mount mounting medium, by Polysciences (1 mentions)
Lsm 710 meta, by Zeiss (1 mentions)
Oil red o, by Merck Group (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Anti rabbit igg conjugated to alexa 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Chicken anti gfp, by Abcam (1 mentions)
Epifluorescence microscope, by Zeiss (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
Rhodamine conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Affinipure donkey anti goat igg, by Jackson ImmunoResearch (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
10 protocols using lsm image browser
1
Immunofluorescence Microscopy Protocol
2
Oil Red O Staining of Drosophila Midguts
3
Dissection and Immunostaining of Drosophila Thorax
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Flies were fixed in 4% formaldehyde with PBS containing 0.3% TritonX-100 for 4 h at 4 °C on a rotator and rinsed with PBS to remove any residual formaldehyde. Fixed flies were then dissected in PBS. Briefly, fly wings were removed carefully without tearing the tissue of the thorax. Then, the head and abdomen together with the intestines were removed so only the thorax would remain. Holding onto the legs as support to stabilize the thorax, a sharp blade was used to make a slice down the middle of the thorax (dorsal side). The thorax was transferred into an Eppendorf tube and washed with PBS to remove any debris. The primary antibodies were used at the following dilutions: mouse anti-ATP5A 1:500 (Abcam Cat# ab14748 RRID: AB_301447) and rabbit anti-Ref2p 1:1000. Alexa 488-conjugated and Alexa 568-conjugated secondary antibodies were used at 1:500. Samples were mounted in Vectashield. Imaging was performed using the LSM710 confocal microscope (Zeiss). Images were processed with the Zeiss LSM Image Browser and Adobe Photoshop.
4
Lipid and Fluorescent Staining Protocol
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To observe neutral lipids stained with Nile red an excitation wavelength of 488 nm, with emission recorded with a 585–615 nm filter set, was used. For polar lipids, an excitation wavelength of 543 nm was used, and emission recorded with a >650 nm filter set. For both CFW and DAPI stained samples, the excitation wavelength was 405 nm with emission measured between 420 and 480 nm. For imaging GFP, a 488 nm excitation wavelength was used with GFP fluorescence detected with a 505–530 nm band pass. All observations were obtained from at least three independent experiments. After acquisition, images were analyzed and processed with LSM Image Browser and Adobe Photoshop (Adobe Systems). Contrast and brightness levels were optimized.
5
Lens Morphometry and Fiber Width
6
Immunostaining of Drosophila Intestines
7
Drosophila Brain Staining and Imaging
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For Drosophila brain staining, dissection of third instar larvae was performed as previously described.32 Briefly, brains attached to the cuticle from the third instar larvae were fixed in 500 µl of 4% formaldehyde for 30 min at room temperature. The fixation solution was discarded and the samples were washed in PBS containing 0.3% Triton X-100. Incubations with primary (overnight at 4°C) and secondary (1 h at room temperature) antibodies were as detailed in Supplementary Table 2 . Samples were mounted in Vectashield (Vector Laboratories). Imaging was performed by LSM710 confocal microscope (Zeiss). Images were processed with the Zeiss LSM Image Browser and Adobe Photoshop.
8
Localization of ENaC Subunits in Epithelial Cells
9
Dissecting and Immunostaining Drosophila Thorax
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Flies were fixed in 4% formaldehyde with PBS containing 0.3% TritonX-100 for 4 hours at 4'C on rotator and rinsed with PBS to remove any residual formaldehyde. Fixed flies were then dissected in PBS. Firstly, fly wings were remove carefully without tearing the tissue of the thorax. Then the head and abdomen together with the intestines are removed so only the thorax would remain. Holding onto the legs as support to stabilize the thorax, a sharp blade was used make a slice down the middle of the thorax (dorsal side). The thorax was transferred into an Eppendorf tube and washed with PBS to remove any debris. The primary antibodies were used at the following dilutions: mouse anti-ATP5A 1:500, rabbit anti-Ref2p 1:1000. Alexa 488 conjugated and Alexa 568 conjugated secondary antibodies were used at 1:500. Samples were mounted in Vectashield. Imaging was performed using LSM710 confocal microscope (Zeiss).
Images were processed with Zeiss LSM Image Browser and Adobe Photoshop.
Images were processed with Zeiss LSM Image Browser and Adobe Photoshop.
10
Drosophila Embryo Immunostaining Protocol
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Embryos were collected on grape juice plates for 24 hours at 37°C. Collected embryos were washed twice with deionized water, and dechorionated with 50 % bleach for 3 minutes. After rinsed thoroughly, embryos were fixed for 30 minutes by 1:1 ratio of Heptane (Sigma Aldrich Cat #246654-1L) and 4 % formaldehyde (Thermo Fisher Cat#F79500) in 1 x Phosphate Buffered Saline (PBS), pH 7.4. To remove vitelline membranes, embryos were shaken vigorously in methanol for 5 times. 1 x PBS, pH 7.4 containing 0.2% BSA and 0.3 % Triton-X100 were used for rehydration and washing. The primary antibodies were used for overnight at the following dilutions: rat anti-Elav 1:500 (DSHB Cat# 7E8A10 RRID:AB_528218), rabbit anti-bgalactosidase 1:250 (Invitrogen Cat# A-11132 RRID: AB_221539), rabbit anti-GFP 1:1000 (Invitrogen Cat# A-11122 RRID:AB_221569), Alexa 647 conjugated goat anti-Horseradish Peroxidase 1:500 (Jackson ImmunoResearch Labs Cat# 123-605-021 RRID: AB_2338967).
Alexa 488 conjugated anti-rat (Invitrogen Cat# A-21208 RRID: AB_2535794), and Alexa 568 conjugated anti-rabbit (Invitrogen Cat# A-11011 RRID: AB_143157) secondary antibodies were used at 1:500. Samples were mounted in Vectashield (Vector Labs Cat# 10198-042, Burlingame, CA). Imaging was performed using LSM710 confocal microscope (Zeiss). Images were processed with Zeiss LSM Image Browser and Adobe Photoshop.
Alexa 488 conjugated anti-rat (Invitrogen Cat# A-21208 RRID: AB_2535794), and Alexa 568 conjugated anti-rabbit (Invitrogen Cat# A-11011 RRID: AB_143157) secondary antibodies were used at 1:500. Samples were mounted in Vectashield (Vector Labs Cat# 10198-042, Burlingame, CA). Imaging was performed using LSM710 confocal microscope (Zeiss). Images were processed with Zeiss LSM Image Browser and Adobe Photoshop.
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