DRG neurons, DRG-Schwann cell or SCG-Schwann cell cultures were rinsed in phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde for 20 minutes. After washing with PBS, samples were permeabilized in ice-cold methanol for 20 minutes and incubated in blocking solution (5% normal donkey-serum, 0.3% Triton X100) for 1hr at room temperature. This was followed by an overnight incubation at 4°C with primary antibodies against MBP and neurofilament M prepared in blocking solution. After washing with PBS, samples were incubated with secondary antibodies for 1hr. Nuclei of cells were visualized by staining with DAPI. Cultures were mounted in antifading agent Citifluor (Ted Pella). Preparations were examined by epifluorescence on a Nikon Eclipse TE2000-U (20x/0.75 objective) microscope. 15 to 20 random field images were acquired per culture using the MetaMorph software package (Molecular Devices). Quantitation was done in ImageJ.
Citifluor
Manufactured by Ted Pella
Citifluor is a mounting medium designed for use in fluorescence microscopy. It is a glycerol-based solution that helps to reduce photobleaching and maintain the fluorescence of labeled samples. Citifluor is suitable for a variety of fluorophores and can be used to mount and preserve specimens for microscopic analysis.
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Lab products found in correlation
Alexa fluor 488 goat anti rabbit antibody, by Thermo Fisher Scientific (3 mentions)
880 light scanning microscope, by Zeiss (2 mentions)
Zen 2.1 acquisition software, by Zeiss (2 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
Metamorph software package, by Molecular Devices (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Tyramide superboost kit, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Trueblack lipofuscin autofluorescence quencher, by Biotium (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Fv 10 microscope, by Olympus (1 mentions)
Microscope slide, by Avantor (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Metamorph imaging software, by Molecular Devices (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Axiocam hrm camera, by Zeiss (1 mentions)
Axioskop fluorescent microscope, by Zeiss (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
12 protocols using citifluor
1
Immunostaining of DRG and Schwann Cells
2
Immunofluorescence Staining of Tetrahymena
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Cells were washed in 10 mM Tris, pH 7.4, and then fixed in 3.2% paraformaldehyde and 0.25% Triton X-100 in PHEM buffer (60 mM 1,4-piperazinediethanesulfonic acid, 25 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid, 10 mM EGTA, and 2 mM MgCl2, pH 6.9) for 10 min at 23°C. Cells were washed twice in 0.1% BSA-PBS, and either dried on a coverslip at 23°C or maintained in Eppendorf tubes for the remainder of the staining protocol. Blocking was performed in 1% BSA-PBS for 20 min, followed by incubation in primary antibody diluted in 1% BSA-PBS for 1 h at 23°C. Primary antibodies used in this study were mouse anti-Kl Ag (1:100; Williams et al., 1990 (link)), rabbit anti-TtCen1 (1:2,000; Stemm-Wolf et al., 2005 (link)), mouse anti-α-tubulin (1:200, 12G10, Developmental Studies Hybridoma Bank, University of Iowa; Wloga et al., 2010 (link)), and rabbit anti-TtSas6A (1:250; Culver et al., 2009 (link)). Cells were then washed twice with 0.1% BSA-PBS before incubation with secondary antibodies diluted in 1% BSA-PBS. Secondary antibodies used in this study were IgG derived from goat and conjugated to Alexa Fluor 488, 594, or 647 (Invitrogen) and diluted to 1:1,000. Hoechst 33342 DNA dye (Sigma-Aldrich) was used at 1:1,000. Cells were mounted in either Citifluor (Ted Pella) or Prolong Gold (Molecular Probes) mounting media. Coverslips were sealed with clear nail polish.
3
Comprehensive Embryonic Lung Immunohistochemistry
4
Quantifying Dopaminergic Neuron Survival
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GFP expression in UAS-GFP/+; th-GAL4/+ organisms were used as a reporter for surviving dopaminergic neurons. Neuronal survival was quantified by dissecting out brains of the corresponding age and treatment. Brains were fixed with 4% formaldehyde in PBS, mounted individually in Citifluor (Ted Pella Inc. Redding, CA.) and images were taken at the National Laboratory of Advanced Microscopy using a confocal Olympus FV10 microscope with a 20X objective. Five brains of each genotype, age and condition were counted in a blinded manner. Confocal acquisition parameters were set and fixed using 10 days old control flies (UAS-GFP/+:Th-GAL4/+). the whole brain was sampled avoiding pixel saturation in the brightest section with optimal pinhole aperture and optimal section thickness, steps were 0.5X section thickness so the whole tissue was sampled twice, after data collection, maximum intensity projections (assembled with image J, https://imagej.nih.gov/ij/ taking in account section thickness and 2X oversampling) were used to make a single flat image and neurons were counted, if there was ambiguity about the number of neurons in a particular cluster in a particular maximally projected brain image, individual sections were analyzed. All other conditions and genotypes were acquired using these pre-set acquisition parameters.
5
Imaging Cellular Proteins in Tetrahymena
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All image collection was conducted at room temperature with an Eclipse Ti inverted microscope (Nikon, Japan) with a CFI Plan Apo VC 60× H numerical aperture 1.4 objective (Nikon, Japan) and a charge-coupled device camera (CoolSNAP HQ2; Photometrics, Tucson, AZ). Image acquisition was through the MetaMorph Imaging Software (Molecular Devices, Sunnyvale, CA).
For live-cell imaging of GFP- and mCherry-tagged proteins, cells were washed in 10 mM Tris-HCl (pH 7.4), pelleted, and placed on microscope slides (VWR, Radnor, PA). For immunofluorescence assay, cells were fixed with 3% formaldehyde, followed by 15% ethanol (68 (link)). Fixed cells were placed on poly-l -lysine-coated multiwell slides (Polysciences, Warrington, PA) and blocked for 1 h with phosphate-buffered saline (PBS) plus 1% bovine serum albumin (BSA) before primary antibody incubation. The Tetrahymena Cen1 antibody was used at 1:2,000 (30 (link)), and antibody incubation was carried out overnight at 4°C. The secondary antibody used for a 2-h room temperature incubation was Alexa Fluor 488 (Invitrogen)-conjugated anti-rabbit antibody at 1:1,000. Primary and secondary antibodies were in PBS plus 1% BSA, and washes between antibody incubations were performed with PBS plus 0.1% BSA. Cells were mounted in CitiFluor (Ted Pella, Redding, CA).
For live-cell imaging of GFP- and mCherry-tagged proteins, cells were washed in 10 mM Tris-HCl (pH 7.4), pelleted, and placed on microscope slides (VWR, Radnor, PA). For immunofluorescence assay, cells were fixed with 3% formaldehyde, followed by 15% ethanol (68 (link)). Fixed cells were placed on poly-
6
Immunofluorescent Localization of GSH
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Thin sections were incubated in phosphate-buffered saline (pH 7.2) containing 2.5% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature and then treated with primary anti-GSH antibody (Agrisera prod. No. AS09 594) followed by secondary antibody (anti-rabbit IgG Alexa549-labelled secondary antibody; Molecular Probes). Negative controls were prepared by omitting the primary antibody. After washing three times, samples were mounted with citifluor (Ted Pella). Fluorescence microscopy observations were performed using a Nikon Eclipse TE2000-U inverted microscope fitted with a ProgRes MF Cool CCD microscope camera (Jenoptik). Fluorescence intensity was quantified using ImageJ software. Data presented are the average of a minimum of nine images obtained in three independent experiments.
7
Synaptotagmin Localization in Drosophila Larvae
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To visualize the localization of transgenic synaptotagmin, third instar larvae were dissected in Ca2+-free HL3.1 saline and fixed in PBS containing 4% formaldehyde for 1 hour. Samples were probed overnight at 4°C in a 1:400 dilution of Dsyt-CL1 in dilution media [PBS with 0.1% Triton (PBST), 1% BSA, and 1% NGS (Jackson ImmunoResearch, West Grove, PA)]. Samples were washed in PBST for 1–3 hours, incubated in dilution media containing a 1:400 dilution of Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen, Carlsbad, CA) for 1 hour at room temperature, washed in PBST for one hour, and mounted on microscope slides in Citifluor (Ted Pella, Redding, CA). Images of neuromuscular junctions of abdominal muscles 6/7 in segments 3 and 4 were acquired using a Zeiss 880 light scanning microscope, a 40X objective, and Zeiss Zen 2.1 acquisition software, version 11,0,3,190.
8
Immunocytochemical Analysis of Microglia
9
Immunohistochemical Analysis of Tyrosine Hydroxylase
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Brains were dissected in PBS, fixed with 4% paraformaldehyde in PBS for 15 min and washed 3 times for 15 min with PBS supplemented with 0.2 % Triton X-100 (PBST). Brains were blocked with 4% goat serum in PBST for 30 min and incubated overnight at 4 °C with anti-TH (Immuno Star 22,941) 1:1000 in PBST supplemented with 4% goat serum. After incubation, brains were washed 3 times for 15 min in PBST and incubated with goat anti-mouse Cy3 1:1000 in 4% goat serum PBST at 4 °C overnight. Washed 3 times for 15 min in PBST and mounted in Citifluor (Ted Pella Inc. Redding Ca). Brains were imaged in the Laboratory of Advanced Microscopy of the Instituto de Biotecnología (UNAM) with an inverted Olympus FV1000 confocal microscope using a 20X objective.
10
Visualizing Synaptotagmin Localization
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The localization of the transgenic synaptotagmin protein was visualized by immunohistochemistry. L3s were dissected in Ca2+-free HL3.1, fixed in PBS containing 4% formaldehyde for 1 hour, incubated with a 1:400 dilution of Dsyt-CL1 in dilution media [PBS with 0.1% Triton (PBST), 1% BSA, and 1% NGS] overnight at 4°C, washed in PBST for 1–3 hours, incubated in dilution media containing a 1:400 dilution of Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen, Carlsbad, CA) for 1 hour at room temperature, washed in PBST for one hour, and mounted on microscope slides in Citifluor (Ted Pella, Redding, CA). Confocal images of the neuromuscular junction on muscle fibers 6 and 7 were taken on a Zeiss 880 light-scanning microscope (Zeiss, White Plains, NY), with a 40x objective and Zeiss Zen 2.1 acquisition software, version 11.0.3.190.
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