Non-expanded (Q30) ataxin-1 and truncated ataxin-1 fusion proteins were constructed in pEYFP or pECFP vectors using the standard PCR and mutagenesis methods previously established in our laboratory (Menon et al., 2012 (link)). COS cells were grown in chamber slides in Dulbecco’s modified Eagle medium supplemented with 10% foetal bovine serum and 100 U/ml penicillin-streptomycin (Invitrogen Life Technologies). Cells were transfected with appropriate plasmid DNA using GeneCellin tranfection reagent (BioCellChallenge). Cells were fixed using 4% paraformaldehyde 54 h post-transfection and slides were mounted using CitiFluor (Agar Scientific). Cells were observed and recorded using a laser scanning confocal microscope (de Chiara et al., 2009 (link)).
Citifluor
Manufactured by Agar Scientific
Sourced in United Kingdom
Citifluor is a mounting medium designed for fluorescence microscopy. It is formulated to reduce photobleaching and maintain the fluorescence of samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Blocking reagent, by Roche (3 mentions)
Sp5 confocal microscope system, by Leica (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Goat α mouse alexa 594, by Thermo Fisher Scientific (2 mentions)
Streptavidin cy3, by Thermo Fisher Scientific (2 mentions)
Neuronal nuclei (neun), by Merck Group (2 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Sp5 laser scanning confocal microscope, by Leica (2 mentions)
Spe confocal microscope, by Leica (2 mentions)
Bx41 light microscope, by Olympus (2 mentions)
Dulbecco s modified, by Thermo Fisher Scientific (1 mentions)
Lsm 510 meta confocal microscope, by Zeiss (1 mentions)
Tcs spii confocal microscope, by Leica (1 mentions)
Ab115513, by Abcam (1 mentions)
Ab8135, by Abcam (1 mentions)
Ab125212, by Abcam (1 mentions)
Ab178847, by Abcam (1 mentions)
30 protocols using citifluor
1
Fluorescent Fusion Protein Visualization
2
Immunofluorescent Localization of CA IX and NCX1
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Cells grown on glass cover slips were fixed in ice-cold methanol for 5 min. Non-specific binding was blocked by incubation with PBS containing 3% BSA for 1 h at 37 °C. Cells were then incubated with primary antibodies for 1 h at 37 °C and washed four times with PBS containing 0.02% Tween 20 for 10 min. The following primary antibodies were used: anti-human CA IX mouse monoclonal antibody M75 in undiluted hybridoma medium [5 (link)], rabbit polyclonal antibody NCX1 (p11-13, Swant, Bellizona, Switzerland) diluted 1:200 in PBS with 1% BSA. After washing the cells were inclubated with Alexa-conjugated secondary antibodies (Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 donkey anti-rabbit IgG) diluted 1:1000 in PBS-BSA for 1 h at 37 °C, and washed four times with PBS. Finally, cells were mounted onto slides in mounting medium with Citifluor (Agar Scientific Ltd., Essex, UK) analyzed by an LSM 510 Meta confocal microscope (Zeiss, Jena, Germany). Images were taken with Plan Neofluar 40×/1.3 oil objective at optical zoom 2 in multi-track mode. The acquired images were processed in ImageJ, the Co-localization. Finder plug-in was used to assess the co-localization of CA IX and NCX1 signals. The background pixels were excluded from the co-localization analysis, the co-localizing pixels were determined and marked in green.
3
Immunofluorescence Analysis of Rat Sciatic Nerve
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Rat sciatic nerves were fixed in situ in 4% PFA for 10 min, dissected, embedded in O.C.T. Compound (Tissue Freezing Medium; Solarbio, Shanghai, China). Sciatic nerve cryosections (5-μm thick) were incubated with acetone for 10 min at − 20 °C, washed in PBS/0.1% Tween 20, blocked for 30 min at room temperature (RT) in blocking buffer (0.3% Triton X-100/10% goat serum/phosphate buffer saline ¼ PBS), and incubated with primary antibodies overnight at 4 °C in blocking buffer. Sections were then washed 3 times in blocking buffer, and sections were incubated with secondary antibodies for 1 h at RT in the dark. Sections were washed again, incubated with DAPI for 5 min at RT, washed and mounted in Citifluor (Agar Scientific).
The primary antibodies used for IF were as follows: neurofilament (1:1000, Abcam, ab8135), SCG10 (1:500, Abcam, ab115513), IBA1 (1:100, Abcam, ab178847), and CD68 (1:100, Abcam, ab125212). All secondary antibodies were also purchased from Abcam. Images were acquired using a Leica TCS SP-II confocal microscope.
The primary antibodies used for IF were as follows: neurofilament (1:1000, Abcam, ab8135), SCG10 (1:500, Abcam, ab115513), IBA1 (1:100, Abcam, ab178847), and CD68 (1:100, Abcam, ab125212). All secondary antibodies were also purchased from Abcam. Images were acquired using a Leica TCS SP-II confocal microscope.
4
Immunohistochemical Analysis of Sciatic Nerve
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Sciatic nerves were dissected out and fixed for 5 h in 4% paraformaldehyde (in PBS, PH7.2) at 4°C. Nerves were then washed in PBS (3 × 10 min) and dehydrated in 30% sucrose (in PBS) overnight at 4°C. Subsequently, nerves were embedded in OCT medium, frozen, and sectioned on a cryostat at a thickness of 12 μm. Sections were permeabilised with 0.25% Triton X-100 plus 1% bovine serum albumin (BSA) in PBS for 45 min and then blocked with blocking buffer (3% BSA plus 0.05% Triton X-100 in PBS) for 1 h at room temperature. Sections were incubated with Jun (Cell Signalling, #9165S), Junb (Cell Signalling, #3753S), Sox2 (Cell Signalling, #2748), Runx2 (Cell Signalling, #8486), or Runx1 (Cell Signalling, #4334) primary antibody (1:100 diluted in blocking buffer) overnight at 4°C. The next day, sections were washed with PBS (3 × 10 min) and then incubated with donkey anti-rabbit secondary antibody conjugated with Alexa Fluor 568 (1:300 diluted in blocking buffer) 1 h at room temperature. Hoechst dye (1:500) was also added into secondary antibody solution in order to label cell nuclei. Finally, sections were washed with PBS (3 × 10 min) and mounted with Citifluor (Agar Scientific, R1320) for imaging with a LeicaSPE confocal microscope.
5
Immunofluorescence Staining of Exogenous and Endogenous Proteins
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293T cells (1 x 105) were grown on coverslips in 6-well tissue culture plates and transiently transfected with plasmids for 24 hr. After transfection, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 30 min, and then permeabilized with 0.1% Triton X-100 in PBS for 7 min. To detect the FLAG-tagged proteins, cells were initially incubated with blocking solution (1% BSA) at room temperature for 1 hr, and then treated with polyclonal rabbit anti-FLAG antibody (F7425; Sigma-Aldrich) for 2 hr. After three washing steps, Alexa Fluor 594-conjugated goat anti-rabbit IgG polyclonal antibody (A11037; Molecular Probes) was added to treat the cell samples. Staining with 4′-6-diamidino-2-phenylindole (DAPI) was performed at room temperature for 15 min. Cells were mounted in Citifluor (Agar Scientific) and observed under a confocal laser-scanning microscope (model LSM 510 META; Zeiss) [37 (link)]. To determine the localization of endogenous ORF50 and MDM2, HH-B2 cells were first treated with 3 mM sodium butyrate (SB). At 6 hr after SB treatment, MG132 was added at a final concentration of 5 μM and the cells were incubated for another 20 hr. Cells were then fixed and stained using antibodies specific to MDM2 (M4308; Sigma-Aldrich) and ORF50 [38 (link)].
6
Immunofluorescence Staining of HeLa and LHCNM2 Cells
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HeLa cells were seeded on poly-d -lysine coated glass slides and fixed 48 h posttransfection. HeLa cells were washed once with PBS (Gibco) and fixed with 2% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature. Cells were washed with PBS and PBS+ (PBS with 0.5% BSA and 0.15% glycine)34 (link),49 (link). Primary antibody incubation was performed overnight at 4 °C in a humid chamber and Alexa-conjugated dyes (Invitrogen, Carlsbad, CA, USA; Alexa Fluor 488 goat-anti-Rabbit, Alexa Fluor 488 donkey-anti-Rabbit, Alexa Fluor 594 goat-anti-Rabbit, Alexa Fluor 594 donkey-anti-Rabbit, Alexa Fluor 488 goat-anti-mouse, Alexa Fluor 594 goat-anti-mouse, and Alexa Fluor 594 chicken-anti-rat) were applied for 1.5 h at room temperature to visualize primary antibodies. Nuclear counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) or Hoechst 33258 for 5 min, and samples were and embedded in glycerol (CitiFluor, Agar Scientific). LHCNM2 cells were grown on poly-l -lysine coated glass coverslip. 24 h after seeding, cells were transfected using lipofectamine 3000 according to manufacturer protocol. After 24 h transfection, cells were fixed with 3.7% formaldehyde for 9 min at room temperature, permeabilized with cold (−20 °C) acetone for 5 min at −20 °C. Cells were rinsed with PBS 1× and processed for immunostaining.
7
Immunofluorescence Staining of Cultured Cells
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Cells were seeded on glass coverslips, and fixed 24 hours later for 10 minutes in 3.7% paraformaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2 pH 6.9) at 37°C. After washing with PBS, cells were permeabilized with 0.1% Triton-X100 for 10 minutes, washed with PBST, and blocked using TNB (100 mM TRIS pH 7.5, 150 mM NaCl, 0.5% Blocking Reagent (Roche)) for 30 minutes. Cells were incubated with primary antibody as indicated, in TNB for one hour. Subsequently cells were washed five times with PBST, and incubated with secondary antibodies (Goat α Mouse Alexa 594 (Invitrogen)) in TNB for one hour. Next, cells were washed five times with PBST and dehydrated using alcohol, prior to embedding them in Citifluor (Agar Scientific) containing 400 ng per μL DAPI (Sigma) and sealing the slides with nail varnish. Images were recorded on a Leica SP5 confocal microscope system using 488 nm and 561 nm lasers for excitation, a 63× lens for magnification, and were analyzed with Leica confocal software.
8
Detailed Reagent Sourcing for Cell Culture
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Eagle's minimal essential medium (EMEM) and l ‐glutamine were obtained from Lonza Group (Basel, Switzerland), penicillin/streptomycin was obtained from Gibco Life Technologies (Paisly, UK), foetal bovine serum (FBS) was obtained from Thermo Scientific (Waltham, MA, USA), bovine serum albumin (BSA) was obtained from Sanquin (Sanquin, Netherlands) and culture plates and chamber slides were obtained from Corning (Corning, NY, USA). ACHP (#4547) was purchased from Tocris (Bristol, UK), recombinant human TGFβ1 (#100‐21) from Peprotech (London, UK), and l ‐ascorbic acid 2‐phosphate magnesium salt (#A‐8960) from Sigma‐Aldrich (St. Louis, MO, USA). FARB buffer and the RNA extraction kit were purchased from Favorgen Biotech (Ping‐Tung, Taiwan), the cDNA synthesis kit was from Fermentas (Vilnius, Lithuania), methanol and acetone was from Merck (Darmstadt, Germany), SYBR Green Master Mix was from Roche (Pleasanton, CA, USA), streptavidin‐CY3 was from Invitrogen (Carlsbad, CA, USA) and Citifluor was from Agar Scientific (Stansted, UK).
9
Immunofluorescence Staining of Brain Cryosections
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The brain cryosections were first incubated with 70% Ethanol for 5 min at room temperature, then washed with PBS and incubated for 40 s with 40 mg/ml Proteinase K, and blocked for 1 h at room temperature in blocking buffer (0.3% Triton X-100/10% Goat serum/phosphate buffer saline), and incubated with primary antibodies overnight at 4 °C in blocking buffer. Sections were then washed three times in blocking buffer and secondary antibodies were incubated for 1 h at room temperature in the dark. Sections were washed again, incubated with DAPI for 5 min at room temperature, washed and mounted in Citifluor (Agar Scientific).
Primary antibodies used for Immunofluorescence were as follow: NeuN (1:1000, ABN78, sigma) All secondary antibodies were also purchased from Abcam.
Primary antibodies used for Immunofluorescence were as follow: NeuN (1:1000, ABN78, sigma) All secondary antibodies were also purchased from Abcam.
10
Fluorescent In Situ Hybridization of Amoeba Cells
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Amoeba cells were harvested by centrifugation (3000 × g, 8 min), washed with ASW and left to adhere on slides for 30 min prior to fixation with 4% formaldehyde (15 min at room temperature). The samples were hybridized for two hours at 46 °C at a formamide concentration of 25% using standard hybridization and washing buffers18 and a combination of the following probes: symbiont specific probes JAMX8_197 (5′-GAAAGGCCAAAACCCCCC-3′) or A1_1033 (5′-GCACCTGTCTCTGCATGT-3′), together with EUK-516 (5′-ACCAGACTTGCCCTCC-3′19 (link)) targeting most eukaryotes and the EUB338 I-III probe mix (5′-GCTGCCTCCCGTAGGAGT-3′, 5′-GCAGCCACCCGTAGGTGT-3′, 5′-GCTGCCACCCGTAGGTGT-3′ 20 (link)) targeting most bacteria. FISH probes were designed based on a multiple 16S rRNA sequence alignment in the software ARB21 (link) using the integrated probe-design tool. Furthermore, thermodynamic parameters and binding specificity were evaluated with the web-based tools mathFISH and probeCheck22 (link)23 (link). All probes were purchased from ThermoFisher Scientific (Germany). Cells were subsequently stained with DAPI (0.5 μg/ml in double distilled water, 3 min), washed once and embedded in Citifluor (Agar-Scientific, UK). Slides were examined using a confocal laser scanning microscope (SP8, Leica, Germany).
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