Cells were fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich, St. Louis, MO), washed three times with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 (Sigma-Aldrich), and incubated in blocking buffer (0.1% Triton X-100) and 5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 1 h at room temperature. The cells were then incubated with primary antibodies in blocking buffer overnight at 4 °C, then stained with compatible Alexa 488- or Alexa 555-conjugated secondary antibodies (Thermo Fisher Scientific) in PBS for 30 min at room temperature. The antibodies are described in Additional file 1 : Table S2.
Alexa 488 or alexa 555 conjugated secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa 488- or Alexa 555-conjugated secondary antibodies are fluorescent-labeled antibodies designed for use in immunoassays and other applications. These antibodies specifically bind to the primary antibody, allowing for the detection and visualization of target proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 confocal microscope, by Zeiss (6 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Vectashield mounting medium containing dapi, by Vector Laboratories (1 mentions)
Zen 2012, by Zeiss (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Cytospin centrifuge, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Eclipse e600 microscope, by Nikon (1 mentions)
Leucosep tube, by Greiner (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Anti v5 tag, by Thermo Fisher Scientific (1 mentions)
Mouse anti rfp, by Abcam (1 mentions)
Alexa 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Tom20, by Santa Cruz Biotechnology (1 mentions)
Ab1542, by Abcam (1 mentions)
Lsm 880 nlo, by Zeiss (1 mentions)
C11 bodipy581 591, by Thermo Fisher Scientific (1 mentions)
10 protocols using alexa 488 or alexa 555 conjugated secondary antibody
1
Immunofluorescence Staining of Fixed Cells
2
BeWo Cell Fusion Assay with Tg-CM
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BeWo cells were grown on coverslips cultured for 24 h, after which cells were treated with Tg-CM or CRT-depleted Tg-CM for 24 h at 37 °C in the presence of forskolin (80 μM). Cells were then fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized with 0.5% Triton X-100 in PBS for 10 min. After the cells were washed 3 times with PBS, they were blocked with 3% bovine serum albumin in PBS for 30 min and incubated with primary antibodies followed by Alexa 488- or Alexa 555-conjugated secondary antibodies (Thermo Fisher Scientific). Stained specimens were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA) and examined with a LSM700 laser scanning confocal microscopy and the LSM software ZEN 2012 (Carl Zeiss, Jena, Germany). For determination of fusion indexes, numbers of nuclei in STB-like fused cells and the total numbers of nuclei were counted in 8 to 10 randomly selected microscopic fields in each E-cadherin-stained specimen. Fusion indexes were calculated as the percentages of the total number of nuclei in the STB-like fused cells to the total number of nuclei in the microscopic fields.
3
Immunocytochemical Analysis of Cells
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For immunocytochemical analyses, the cells were fixed with 4% paraformaldehyde. After boiling in 0.1 mM citrate buffer (pH 6.0) for 1 h and blocking in blocking buffer (phosphate-buffered saline [PBS] containing 10% normal donkey serum) for 1 h at room temperature, the cells were incubated with primary antibodies at 4 °C overnight. After three washes with PBS, the cells were incubated with Alexa 488- or Alexa 555-conjugated secondary antibodies (Life Technologies) for 1 h at room temperature. The nuclei were stained with 10 μg/mL Hoechst 33258 (Sigma). After washing with PBS, the cells were examined using a confocal laser scanning microscope (LSM700; Carl Zeiss).
4
Quantification of DNA Double-Strand Breaks
5
Immunofluorescence Analysis of Ovarian Tissue
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Ovaries were dissected in PBS, and fixed for 1 h at room temperature in 4% paraformaldehyde in PBS under shaking. Tissues were subsequently rinsed in PBS with 0.1% Triton X-100 (PBT), permeabilized for 1 h in PBT with shaking, and blocked in 2% bovine serum albumin in PBT for 1 h with shaking. Tissues were incubated with primary antibodies in 2% bovine serum albumin in PBT overnight at 4 °C with shaking. After 1-h washing in PBT, secondary antibodies and DAPI were applied at room temperature for 2 h and washed for 1 h in PBT, and the tissues were mounted in fluorescence mounting medium (Dako). Primary antibodies are as follows: mouse anti-RFP (Abcam, #ab62341, 1:500), rabbit serum anti-MSRO [(13 ), 1:300], and anti-V5 tag (Thermo Fisher Scientific, #25F11F7, 1:500). Alexa488- or Alexa555-conjugated secondary antibodies (Life Technologies) were used at 1:1,000. Alexa647 Phalloidin (Life Technologies) was used to stain F-actin (1:200). All images were taken on a Zeiss LSM 700 confocal microscope and processed using ImageJ.
6
Quantifying adipose tissue innervation
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The inguinal fat pad of fed mice was fixed in paraformaldehyde 4% overnight before being cut into 300 µm sections using a vibratome (Campden). Sections were incubated in blocking solution (2% normal horse serum and 0.2% triton X-100 in PBS) at room temperature before being incubated for 24 h at room temperature with primary UCP1 (1:5000, kindly provided by D. Ricquier), TOM20 (1:400, Santa Cruz, sc-11415) or TH (1:750, Abcam AB1542) antibodies. After overnight incubation at 4 °C with Alexa 488- or Alexa 555-conjugated secondary antibodies (Life Technology), imaging was performed using a confocal laser scanning microscope (LSM880 NLO, Carl Zeiss). Quantification of sympathetic innervation and vascularization was performed on binarized images obtained after TH or lectin labelling. At least ten regions of interest (ROI) of the same size were randomly selected in areas 1, 2 and 3 from one inguinal fat pad based on tissue autofluorescence. Sympathetic nerve or vascular density was expressed as the ratio of, respectively, TH- or lectin-labelled pixels to the total number of pixels per ROI by using Fiji software.
7
Tissue Fixation, Staining, and Lipid Peroxidation Analysis
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Tissues were dissected in PBS and fixed for at least 1 h at room temperature in 4% paraformaldehyde in PBS. Tissues were subsequently rinsed in PBS+0.1% Triton X-100 (PBT), permeabilized and blocked in 2% bovine serum albumin PBT for 1 h, and incubated with first antibody (anti-GFP antibody (1:200, Abcam) or 4-HNE protein adducts antibody (1:100, Abcam)) diluted in blocking solution overnight at 4 C. After extensive washing, Alexa 488-or Alexa 555-conjugated secondary antibodies (Life Technologies) and DAPI (Sigma) were applied at room temperature for 2 h. After extensive washes in PBT, the samples were mounted in Dako Fluorescence Mounting Medium (Agilent). All the images were taken on a Zeiss LSM 700 confocal microscope and processed using FIJI.
Malondialdehyde (MDA) quantification and C11-BODIPY 581/591 staining Hemolymph of 150 female flies was collected by centrifugation as described above. The samples were subjected to the lipid peroxidation (MDA) assay kit (Abcam) according to the manufacturer's instructions. The MDA contents were normalized to protein amount.
To detect lipid peroxidation in Malpighian tubules, flies were injected with 2 mM C11-BODIPY 581/591 (Life technologies). Samples were collected, washed and mounted in Schneider's medium. Images were taken by confocal microscopy and processed by FIJI.
Malondialdehyde (MDA) quantification and C11-BODIPY 581/591 staining Hemolymph of 150 female flies was collected by centrifugation as described above. The samples were subjected to the lipid peroxidation (MDA) assay kit (Abcam) according to the manufacturer's instructions. The MDA contents were normalized to protein amount.
To detect lipid peroxidation in Malpighian tubules, flies were injected with 2 mM C11-BODIPY 581/591 (Life technologies). Samples were collected, washed and mounted in Schneider's medium. Images were taken by confocal microscopy and processed by FIJI.
8
Immunofluorescence Staining of Stem Cells
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Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 for 15 min, and blocked with 5% BSA in PBS for 30 min at room temperature. After blocking, cells were stained with anti-HA antibodies (Santa Cruz), anti-Oct4 antibodies (ab19857, Abcam), or anti-SSEA-1 antibodies (Miltenyi Biotec), or anti-Nanog antibodies (ReproCELL Inc) followed by Alexa 488 or Alexa 555 conjugated secondary antibodies (Life Technologies) and counterstained with Hoechst 33342 (Life Technologies). Images were acquired with Zeiss LSM700 confocal microscope equipped with a ×20 objective lens under the Zen 2010 operating software.
9
Immunostaining of Frataxin-Silenced Cells
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Immunostaining was performed on normal and frataxin-silenced cells cultured on coverslips fixed at room temperature for 10 min with PBS containing 2% paraformaldehyde (PFA) followed by another 10 min with PBS 4% PFA. After several washes with PBS, the cells were permeabilised in PBS containing 0.1% Triton X-100 and 1% BSA (PBS-TS). The cells were then incubated overnight at 4 • C with the primary antibody diluted in PBS-TS and, after washing with PBS, they were incubated for 1 h at room temperature in PBS-TS with the appropriate Alexa-488-or Alexa-555-conjugated secondary antibody (1:1000; Invitrogen-Life Technologies). Following extensive washes, the preparations were stained with 4 ′ -6-diamidine-2-phenylindole (DAPI; 1:5000; Merck-Millipore) and, after washing, the cells were immediately mounted with Fluoromount-G (Southern Biotech Assoc. Inc., Birmingham, AL, US). The labelled preparations were examined on an inverted Axiovert 200 (Zeiss) microscope coupled to a CCD camera (Spot ST, Slider). The antibodies used in the immunohistochemistry assay were monoclonal antibodies against MAP2, clone HM-2, (1:1000, Sigma) and Smi-31 (1:1000; Covance).
10
Immunohistochemical Analysis of α-Synuclein
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The mice were perfused transcardially with phosphate-buffered saline (PBS). Brains were separated and soaked in 4% paraformaldehyde for 48 h. Then, 30% sucrose solution was used for dehydration treatment. A cryostat (Leica SM 2010R, Germany) was used to cut the brains into continuous slices with a thickness of 40 μm. The following primary and secondary antibodies were used as recommended by the manufacturers: rabbit anti-human/mouse α-synuclein (α-syn; Santa Cruz Biotechnology, United States, sc-7011-R, 1:1000), mouse anti-PV (Sigma-Aldrich, United States, P3088, 1:500), rabbit anti-PV (Abcam, United States, ab11427, 1:1000), rabbit anti-c-fos (Sigma-Aldrich, United States, F7799, 1:1000), mouse anti-neuronal nucleus (NeuN; Chemicon, United States, MAB 377, 1:500), and Alexa 488 or Alexa 555 conjugated secondary antibody (Invitrogen, United States, 1:500). For antibodies produced in mice, the mouse-on-mouse immunodetection kit (Vector Laboratories, United States, BMK-2202) was used following the manufacturer’s protocol. A laser scanning confocal microscope (LSM 710; Zeiss, Germany) was used for observing and photographing. The paired images in all the figures were acquired under the same conditions and processed uniformly after collection.
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