Cells were seeded in a cell culture-treated 96-well plate and incubated for 24 h at 37 °C and 5% CO2 to allow for cell attachment. The cell medium was replaced with complete DMEM, and the plate lid was replaced with the AC-modified lid. Cells were incubated at 25 °C for 4, 24 or 48 h with electrical stimulation at 300 kHz and 3 V/ cm in line with previous work (Jain et al. 2022 (link)). The input signal was a sinusoidal wave with a frequency of 300 kHz, an amplitude of 3 V/cm, and an impedance of 50 Ω. The signal was applied without any offset or phase shift. After stimulation for the desired length of time, cell media was replaced with 10% PrestoBlue™ cell viability agent from ThermoFisher Scientific (Waltham, MA, USA) and incubated at 25 °C for 3 h before being transferred to an opaque black 96-well plate (Nunc™; ThermoFisher Scientific (Waltham, MA, USA)). The fluorescence was read using a TECAN Infinite 2000 plate reader (Männedorf, ZH, CHE)) at an excitation wavelength of 560 nm and emission of 590 nm. The data was normalised against a negative control of cells without electrical stimulation and a positive control of cells without stimulation treated with 3% TX-100 from Sigma Aldrich (St. Louis, MO, USA)). This is recorded in all figures as the ‘Relative change in metabolism (%)’.
Tx 100
Manufactured by Merck Group
Sourced in United States, Germany, Switzerland, Poland, Spain
About the product
TX-100 is a non-ionic detergent commonly used in biochemical applications. It is an effective solubilizing agent for membrane proteins and has been widely used in cell lysis, protein purification, and other laboratory procedures.
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Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (8 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (8 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (4 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (4 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (4 mentions)
Triton x 100, by Merck Group (3 mentions)
Vectashield, by Vector Laboratories (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (3 mentions)
Tween 20, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Goat serum, by Thermo Fisher Scientific (3 mentions)
Schneider s insect medium, by Merck Group (3 mentions)
Glycine, by Merck Group (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
Check compatibility
Market Availability & Pricing
Here is the cleaned and reformatted content for display on a product detail page:
Triton X-100 (TX-100) is a non-ionic surfactant commonly used in various laboratory applications. It is a clear, viscous liquid that is soluble in water and other organic solvents. TX-100 is primarily used as a detergent, emulsifier, and solubilizing agent in a variety of scientific and industrial processes.
TX-100 is commercially available from Sigma-Aldrich. However, due to environmental concerns, the European Union has listed TX-100 and related molecules on the REACH Annex XIV, leading to the need for alternatives in various industries. As a result, the continued use of TX-100 for established production processes beyond the "sunset date" (January 2021) is possible to some extent by applying for authorization for continued use for a limited time.
Several alternatives to TX-100 have been developed, such as TX-100 reduced and Nereid, which have been shown to possess high antiviral potency. These alternatives may be recommended as replacements for TX-100 in certain applications.
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148 protocols using «tx 100»
1
Electrical Stimulation and Cell Viability
2
Quantifying TDP-43 Subcellular Localization in iMNs
When iMN differentiation was on Day 13, 96-well plates (for viability assays) and 24-well plates with coverslips (for imaging) were coated overnight at 37°C in Matrigel (Corning). On Day 14 of differentiation iMNs were plated at a density of 30,000 cells/well for 96-well plates and 150,000 cells/well for 24-well plates in Neurobasal media + NEAA, Glutamax, N2, B27 (Gibco), plus 10 ng/mL BDNF, GDNF, CNTF (PeproTech) and 0.2 μg/ml Ascorbic acid (Sigma-Aldrich). iMNs were fed every 2 days and maintained for 13 additional days in the same media. Treatments started on day 13 after plating (DIV27) and were kept on for 24 h.
For viability studies, a VX-745 dose-response analysis was performed to determine a concentration that would not reduce viability significantly in both control and C9 neurons. VX-745 (Tocris) was dissolved in DMSO (Sigma-Aldrich), serial dilutions were made in OptiMEM (Gibco), added dropwise to each well and incubated at 37°C for 24 h iMN viability was measured using the CellTiter-Glo kit (Promega) on 3 separate experiments (n = 3 per run, n = 9 final). Using this approach, we established that 1μM VX-745 exhibited no toxicity.
For imaging and Nuclear-Cytoplasmic distribution of TDP-43 (Nuc/Cyt ratio), VX-745 was used at a concentration of 1μM. Immediately after the 24 h were completed, iMNs were washed once in PBS (Gibco) and fixed in 4% PFA for 20 min (Electron Microscopy Sciences). iMNs were then washed 3 times in PBS and blocked with 5% Donkey Serum (Jackson ImmunoResearch) + 0.3% TX-100 (Sigma-Aldrich) in PBS for 30 min at room temperature. Primary antibodies (goat MAP2 1:1000, Phosphosolutions; rabbit TDP-43 N-terminal 1:300, Proteintech) were diluted in blocking solution and incubated overnight at 4°C. Secondary antibodies (donkey Alexa Fluor, Jackson ImmunoResearch) were used at 1:1000 dilution in blocking solution and incubated for 60 min at room temperature. All treatments/iMN lines were treated and probed simultaneously to decrease variability. Coverslips were mounted in Prolong Glass (Invitrogen) and were left to air dry at room temperature for at least overnight before imaging.
Images were acquired (10/group) using an A1R Nikon Confocal Microscope and fields of view (FOV) were processed for analyses using Nikon NIS Elements Software. Briefly, images were batch processed for Max intensity projection using the GA3 tool. Then, nuclei were selected using the autodetect tool in the ROI editor. Cell bodies (cytoplasm) were hand drawn using the Bezier tool. Data was exported to Excel and nuclear-cytosolic ratios were calculated using the mean intensity values for the 488 channel (TDP-43 signal) for matched nuclear and cytoplasmic selections. Data were analyzed by two-way ANOVA using genotype and treatment as variables using Prism.
For viability studies, a VX-745 dose-response analysis was performed to determine a concentration that would not reduce viability significantly in both control and C9 neurons. VX-745 (Tocris) was dissolved in DMSO (Sigma-Aldrich), serial dilutions were made in OptiMEM (Gibco), added dropwise to each well and incubated at 37°C for 24 h iMN viability was measured using the CellTiter-Glo kit (Promega) on 3 separate experiments (n = 3 per run, n = 9 final). Using this approach, we established that 1μM VX-745 exhibited no toxicity.
For imaging and Nuclear-Cytoplasmic distribution of TDP-43 (Nuc/Cyt ratio), VX-745 was used at a concentration of 1μM. Immediately after the 24 h were completed, iMNs were washed once in PBS (Gibco) and fixed in 4% PFA for 20 min (Electron Microscopy Sciences). iMNs were then washed 3 times in PBS and blocked with 5% Donkey Serum (Jackson ImmunoResearch) + 0.3% TX-100 (Sigma-Aldrich) in PBS for 30 min at room temperature. Primary antibodies (goat MAP2 1:1000, Phosphosolutions; rabbit TDP-43 N-terminal 1:300, Proteintech) were diluted in blocking solution and incubated overnight at 4°C. Secondary antibodies (donkey Alexa Fluor, Jackson ImmunoResearch) were used at 1:1000 dilution in blocking solution and incubated for 60 min at room temperature. All treatments/iMN lines were treated and probed simultaneously to decrease variability. Coverslips were mounted in Prolong Glass (Invitrogen) and were left to air dry at room temperature for at least overnight before imaging.
Images were acquired (10/group) using an A1R Nikon Confocal Microscope and fields of view (FOV) were processed for analyses using Nikon NIS Elements Software. Briefly, images were batch processed for Max intensity projection using the GA3 tool. Then, nuclei were selected using the autodetect tool in the ROI editor. Cell bodies (cytoplasm) were hand drawn using the Bezier tool. Data was exported to Excel and nuclear-cytosolic ratios were calculated using the mean intensity values for the 488 channel (TDP-43 signal) for matched nuclear and cytoplasmic selections. Data were analyzed by two-way ANOVA using genotype and treatment as variables using Prism.
3
Generating Transient Knockout Cells for Mass Spectrometry
To generate transient KO cells of the different genes identified by mass spectrometry, guide RNAs targeting the gene of interest were cloned into pKLV-U6gRNA (BbsI)-PGKblast2ABFP. Viral particles were produced with Lenti-X 293T cells as described above. The HeLa Cas9 stable cell line was then transduced. Briefly, 1 × 104 HeLa Cas9 cells were infected with 250 µl of lentiviral supernatant in a 96-well plate. Seventy-two hours posttransduction cells were replated in a 48-well plate and incubated for an extra 2 d. On d 6, 2 × 104 of transduced cells were plated onto Matrigel-coated (1:100 in complete DMEM; 354277; Corning) glass coverslips (400-03-19; Academy). On d 8, cells were fixed with 4% paraformaldehyde for 15 min. Cells were permeabilized with 0.1% TX-100 (T9284; Sigma-Aldrich) and incubated for 1 h with a blocking solution containing 3% BSA (BP9703-100; Sigma-Aldrich). Cells were incubated for 1 h with anti-ACBD3 and anti-GM130 primary antibodies followed by 1 h incubation with appropriate Alexa Fluor Dyes. PBS1X was used to wash cells in-between all steps and DAPI stained (300 nM; D21490 Invitrogen) for 5 min. Coverslips were mounted in ProLong Gold (P36930; Life Technologies).
4
Primary Skin Mast Cell Degranulation Assay
Release of β-hexosaminidase from LAD2 cells was followed as a measurement of mast cell degranulation using a method described previously (4 (link)).
For human primary skin mast cell degranulation experiments, both β-hexosaminidase and tryptase were measured. Following isolation from primary human skin tissue, CD117+ cells were washed once in relevant assay buffer [β-hexosaminidase as above, and, for tryptase, HBSS (Sigma), both supplemented with 0.3% BSA] and then seeded into a conical bottom 96 well plate at 5 × 104 cells per well. Cells were pre-treated with Compound B or a matched 0.2% DMSO (Sigma) vehicle in assay buffer and incubated at 37°C in 5% CO2 for 30 min. Cells were centrifuged (400g for 10 min), supernatant was removed, and appropriate treatments were added. Cells were incubated at 37°C in 5% CO2 for 1 h. Plates were centrifuged, with supernatants collected and lysates generated [cell pellets lysed in 0.1% Tx100 (Sigma), in assay buffer], and then stored at −80°C for later quantification. β-Hexosaminidase activity assay was performed as already described. For tryptase quantification: The amount of total tryptase (α and β tryptase) was quantified using the ImmunoCap Tryptase Test ran on either a Phadia 100 or 250 Immunoassay Analyzer as per the manufacturer’s protocol. Supernatants and lysates (reference wells were generated for each experiment) were diluted in sample diluent so that they could be interpolated from the standard curve.
For human primary skin mast cell degranulation experiments, both β-hexosaminidase and tryptase were measured. Following isolation from primary human skin tissue, CD117+ cells were washed once in relevant assay buffer [β-hexosaminidase as above, and, for tryptase, HBSS (Sigma), both supplemented with 0.3% BSA] and then seeded into a conical bottom 96 well plate at 5 × 104 cells per well. Cells were pre-treated with Compound B or a matched 0.2% DMSO (Sigma) vehicle in assay buffer and incubated at 37°C in 5% CO2 for 30 min. Cells were centrifuged (400g for 10 min), supernatant was removed, and appropriate treatments were added. Cells were incubated at 37°C in 5% CO2 for 1 h. Plates were centrifuged, with supernatants collected and lysates generated [cell pellets lysed in 0.1% Tx100 (Sigma), in assay buffer], and then stored at −80°C for later quantification. β-Hexosaminidase activity assay was performed as already described. For tryptase quantification: The amount of total tryptase (α and β tryptase) was quantified using the ImmunoCap Tryptase Test ran on either a Phadia 100 or 250 Immunoassay Analyzer as per the manufacturer’s protocol. Supernatants and lysates (reference wells were generated for each experiment) were diluted in sample diluent so that they could be interpolated from the standard curve.
5
Testis and Germ Cell Lysis Protocol
Testis lysates were prepared using RIPA Lysis Buffer (cat # 20-188, Millipore, Temecula, CA), following the Abcam tissue lysate preparation protocol. In brief, one frozen testis was homogenized in 200 μL RIPA buffer containing 0.1% SDS, protease inhibitor cocktail (cat # 05892791001, Roche Diagnostics GmbH, Mannheim, Germany), and phosphatase inhibitor (cat # 04906845001, Roche Diagnostics). The lysate was continuously agitated using a pellet pestle (cat # 749521-1590, Kimble, Millville, NJ) for 5 min, incubated for 30 min on ice, and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was transferred to a fresh tube, and protein was determined using the Bradford-based colorimetric assay (cat # 500-0006, Bio-Rad Protein assay, Bio-Rad, Hercules, CA). The lysate was frozen at −80°C.
Frozen germ cells were lysed in 75 μL buffer (1% IGEPAL (cat #I3021, Sigma-Aldrich), 1% TX-100 (cat #T9284, Sigma-Aldrich), 0.5% deoxycholate (cat #D6750, Sigma-Aldrich), 1.5× phosphatase inhibitor (Roche), and Roche protease inhibitor cocktail dissolved in double-distilled water. The lysate was incubated for 30 min on ice, followed by centrifugation for 5 min at 5,000 g at 4°C. The supernatant was transferred to a fresh tube, and protein quantified using the Bradford-based colorimetric assay. Germ cell lysate was stored at −80°C.
For the detection of MGAT2, 60 μg of protein was separated by 10% SDS-PAGE. Following transfer to a polyvinylidene fluoride (PVDF) membrane in transfer buffer containing 10% methanol for 90 min, the membrane was blocked in TBS Odyssey blocking buffer (TOBB, cat #P/N 927-600001, LICORbio™) for 1 h at 37°C and incubated overnight at 4°C with anti-MGAT2 primary Ab (cat # LS-C40495, LS Bio, Lynnwood, WA) diluted 1:400 in TBS blocking buffer. Secondary Ab, which was donkey anti-rabbit IRDye® 800CW (LICORbio™) at a dilution of 1:8000 in TBS blocking buffer, was applied for 1 h at RT, followed by four washes with TBST (TBS and with 0.05% Tween 20 (cat #P7949, Sigma-Aldrich). Imaging was performed using a LI-COR Odyssey scanner.
For the detection of AKT and ERK, 60 μg of the germ cell extract was electrophoresed by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked for 2 h at RT using TOBB, followed by overnight incubation at 4°C with primary Ab in TOBB. Antibodies obtained from cell signaling technology (Danvers, MA) were the following: rabbit monoclonal antibody (mAb) D13.14.4E targeting pERK1/2 (cat # 4370), mouse mAb 3A7 targeting ERK1/2 (cat # 9107), mouse mAb 40D4 targeting AKT (cat # 2920), and rabbit mAb D9E targeting pAKT (cat # 4060). The secondary Abs used were donkey anti-rabbit IgG IRDye 800CW (cat #P/N926-32213, LI-COR) and goat anti-mouse IgG DyLight™ 680 (cat # 35519, Invitrogen). After incubation, membranes were rinsed four times in TBST and incubated in LI-COR secondary Abs in TOBB for 1 h at RT in the dark. Subsequent washes were four times with TBST and twice with TBS. Imaging was performed using the LI-COR Odyssey Fc imaging system, and band intensity determination was obtained using LI-COR® Acquisition Software (LICORbio™).
Frozen germ cells were lysed in 75 μL buffer (1% IGEPAL (cat #I3021, Sigma-Aldrich), 1% TX-100 (cat #T9284, Sigma-Aldrich), 0.5% deoxycholate (cat #D6750, Sigma-Aldrich), 1.5× phosphatase inhibitor (Roche), and Roche protease inhibitor cocktail dissolved in double-distilled water. The lysate was incubated for 30 min on ice, followed by centrifugation for 5 min at 5,000 g at 4°C. The supernatant was transferred to a fresh tube, and protein quantified using the Bradford-based colorimetric assay. Germ cell lysate was stored at −80°C.
For the detection of MGAT2, 60 μg of protein was separated by 10% SDS-PAGE. Following transfer to a polyvinylidene fluoride (PVDF) membrane in transfer buffer containing 10% methanol for 90 min, the membrane was blocked in TBS Odyssey blocking buffer (TOBB, cat #P/N 927-600001, LICORbio™) for 1 h at 37°C and incubated overnight at 4°C with anti-MGAT2 primary Ab (cat # LS-C40495, LS Bio, Lynnwood, WA) diluted 1:400 in TBS blocking buffer. Secondary Ab, which was donkey anti-rabbit IRDye® 800CW (LICORbio™) at a dilution of 1:8000 in TBS blocking buffer, was applied for 1 h at RT, followed by four washes with TBST (TBS and with 0.05% Tween 20 (cat #P7949, Sigma-Aldrich). Imaging was performed using a LI-COR Odyssey scanner.
For the detection of AKT and ERK, 60 μg of the germ cell extract was electrophoresed by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked for 2 h at RT using TOBB, followed by overnight incubation at 4°C with primary Ab in TOBB. Antibodies obtained from cell signaling technology (Danvers, MA) were the following: rabbit monoclonal antibody (mAb) D13.14.4E targeting pERK1/2 (cat # 4370), mouse mAb 3A7 targeting ERK1/2 (cat # 9107), mouse mAb 40D4 targeting AKT (cat # 2920), and rabbit mAb D9E targeting pAKT (cat # 4060). The secondary Abs used were donkey anti-rabbit IgG IRDye 800CW (cat #P/N926-32213, LI-COR) and goat anti-mouse IgG DyLight™ 680 (cat # 35519, Invitrogen). After incubation, membranes were rinsed four times in TBST and incubated in LI-COR secondary Abs in TOBB for 1 h at RT in the dark. Subsequent washes were four times with TBST and twice with TBS. Imaging was performed using the LI-COR Odyssey Fc imaging system, and band intensity determination was obtained using LI-COR® Acquisition Software (LICORbio™).
Top 5 protocols citing «tx 100»
1
Immunofluorescent Labeling of Neuronal Nuclei
2
Immunofluorescence Analysis of Microglia and Astrocytes
3
Liver Decellularization Protocol
The perfusion regime for the decellularization of the liver left lobe is shown in Table1. The decellularization of the whole liver was achieved by repeating the procedure shown in Table 1 three times (the freezing/thawing step was performed just once). The following abbreviations were used: distilled Water (dH2O), TX100 (Triton X100), SDS (sodium dodecyl sulfate), A-A 5% (Antibiotic and Antimycotic), PAA (paracetic acid) and EtOH (ethanol) purchased from Sigma Aldrich.
The initial flow rate for decellularization perfusion was 0.2–0.3 ml/min/g of liver. Subsequently, two phases of perfusion were adopted a) steeply increasing flow rate to compensate reduced resistance and b) stabilization of the flow rate as the decellularization proceeds. The two phases of flow rate are shown inFig. 8 . After decellularization, 125 mm3 cubic scaffold fragments (5 mm×5 mm×5 mm) were dissected by scalpel cleavage for further characterization and liver bioengineering in vitro.
The initial flow rate for decellularization perfusion was 0.2–0.3 ml/min/g of liver. Subsequently, two phases of perfusion were adopted a) steeply increasing flow rate to compensate reduced resistance and b) stabilization of the flow rate as the decellularization proceeds. The two phases of flow rate are shown in
4
Drosophila Brain Immunohistochemistry Protocol
Visualization of split-GAL4 driver line expression patterns with pJFRC51-3XUAS-IVS-Syt::smHA in su(Hw)attP1 and pJFRC225-5XUAS-IVS-myr::smFLAG in VK00005 (Nern et al., 2015 (link)) or, in a few cases, 20XUAS-CsChrimson-mVenus in attP18 (Klapoetke et al., 2014 (link)) as reporters was performed as described (Aso et al., 2014 (link); Wu et al., 2016 (link)). Detailed protocols are also available online (https://www.janelia.org/project-team/flylight/protocols under ‘IHC - Anti-GFP’, ‘IHC - Polarity Sequential’ and ‘DPX mounting’). Multicolor Flp-out (MCFO) markers were detected by immunolabeling with antibodies against HA, FLAG and V5 epitopes as described (Nern et al., 2015 (link)). Detailed protocols are also available online (https://www.janelia.org/project-team/flylight/protocols under ‘IHC - MCFO’.
For other experiments, brains of female flies were dissected in insect cell culture medium (Schneider’s Insect Medium, Sigma Aldrich, #S0146) and fixed with 2% PFA (w/v) (prepared from a 20% stock solution, Electron Microscopy Sciences: 15713) also in cell culture medium for 1 hr at room temperature. Brains were washed with 0.5% (v/v) TX-100 (Sigma Aldrich: X100) in PBS and incubated in PBT-NGS (5% Goat Serum [ThermoFisher: 16210–064] in PBT) for at least 30 min. Incubations with primary antibodies and subsequently, after additional PBT washes, secondary antibodies, were in PBT-NGS at 4°C overnight. After additional washes with PBT and then PBS, brains were mounted in SlowFadeGold (ThermoFisher: S36937) and imaged on a Zeiss LSM 710 confocal microscope using 20 × 0.8 NA, 40x NA 1.3 or 63 × 1.4 NA objectives. A few specimens were mounted in DPX following the protocol described in Nern et al. (2015) (link). For experiments using only native fluorescence, brains were fixed as above and mounted and imaged after the initial post-fixation washes.
Primary antibodies used in each experiment are indicated inSupplementary file 1E . Primary antibodies were anti-GFP rabbit polyclonal (ThermoFisher: A-11122, RRID:AB_221569 ; used at 1:1000 dilution), anti-GFP mouse monoclonal 3E6 (ThermoFisher: A-11120, RRID:AB_221568 ; dilution 1:100), anti-dsRed rabbit polyclonal (Clontech Laboratories, Inc.: 632496, RRID:AB_10013483 ; dilution 1:1000), anti-HA rabbit monoclonal C29F4 (Cell Signaling Technologies: 3724S, RRID:AB_1549585 ; dilution 1:300), anti-FLAG rat monoclonal (DYKDDDDK Epitope Tag Antibody [L5], Novus Biologicals: NBP1-06712, RRID:AB_1625981 ; 1:200), DyLight 549 or DyLight 550 conjugated anti-V5 mouse monoclonals (AbD Serotec: MCA1360D549GA or MCA1360D550GA, RRID:AB_10850329 or RRID:AB_2687576 ; 1:500 dilution), anti-cockroach allatostatin (Ast7) mouse monoclonal 5F10 (Stay et al., 1992 (link)) (also detects Drosophila AstA (Hergarden et al., 2012 (link)); Developmental Studies Hybridoma Bank (DSHB): RRID:AB_528076 ; dilution 1:5), anti-CadN rat monoclonal DN-Ex #8 (DSHB: RRID:AB_528121 ; dilution 1:20) (Iwai et al., 1997 (link)), anti-chaoptin mouse monoclonal 24B10 (DSHB: RRID:AB_528161 , dilution 1:20. Fujita et al., 1982 (link)) and anti-Brp mouse monoclonal nc82 (Wagh et al., 2006 (link)) (DSHB: RRID:AB_2314866 ; dilution 1:30).
Secondary antibodies (all from Jackson ImmunoResearch Laboratories, Inc) were DyLight 488-AffiniPure Donkey Anti-Mouse IgG (H+L): 715-485-151, 1:500 dilution; DyLight 594 AffiniPure Donkey anti Rabbit IgG (H+L): 711-515-152, 1:300 dilution; Alexa Fluor 647 AffiniPure Donkey Anti-Rat IgG (H+L): 712-605-153, 1:300 dilution; Alexa Fluor 594 AffiniPure Donkey Anti-Mouse IgG (H+L): 715-585-151,1:300 dilution; Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L): 715-605-151, 1:300 dilution and Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L): 711-545-152, 1:1000 dilution.
For other experiments, brains of female flies were dissected in insect cell culture medium (Schneider’s Insect Medium, Sigma Aldrich, #S0146) and fixed with 2% PFA (w/v) (prepared from a 20% stock solution, Electron Microscopy Sciences: 15713) also in cell culture medium for 1 hr at room temperature. Brains were washed with 0.5% (v/v) TX-100 (Sigma Aldrich: X100) in PBS and incubated in PBT-NGS (5% Goat Serum [ThermoFisher: 16210–064] in PBT) for at least 30 min. Incubations with primary antibodies and subsequently, after additional PBT washes, secondary antibodies, were in PBT-NGS at 4°C overnight. After additional washes with PBT and then PBS, brains were mounted in SlowFadeGold (ThermoFisher: S36937) and imaged on a Zeiss LSM 710 confocal microscope using 20 × 0.8 NA, 40x NA 1.3 or 63 × 1.4 NA objectives. A few specimens were mounted in DPX following the protocol described in Nern et al. (2015) (link). For experiments using only native fluorescence, brains were fixed as above and mounted and imaged after the initial post-fixation washes.
Primary antibodies used in each experiment are indicated in
Secondary antibodies (all from Jackson ImmunoResearch Laboratories, Inc) were DyLight 488-AffiniPure Donkey Anti-Mouse IgG (H+L): 715-485-151, 1:500 dilution; DyLight 594 AffiniPure Donkey anti Rabbit IgG (H+L): 711-515-152, 1:300 dilution; Alexa Fluor 647 AffiniPure Donkey Anti-Rat IgG (H+L): 712-605-153, 1:300 dilution; Alexa Fluor 594 AffiniPure Donkey Anti-Mouse IgG (H+L): 715-585-151,1:300 dilution; Alexa Fluor 647 AffiniPure Donkey Anti-Mouse IgG (H+L): 715-605-151, 1:300 dilution and Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L): 711-545-152, 1:1000 dilution.
5
Immunofluorescence Staining of Drosophila Brains
Flies were anesthetized with CO2 to select the desired genotype, washed in cold 70% ethanol (30″), then cold PBS (30″) and kept in cold ExpressFive™ cell culture medium™ (Invitrogen) before and during preparation. Brains were dissected immediately using forceps and transferred into cold fixative. All subsequent steps were performed on a nutator at room temperature in 200 µL PCR tubes.
Brains were fixed in 2% paraformaldehyde (PFA, Electron Microscopy Sciences) in ExpressFive™ medium for 1 h. After three or more washes (15′ each) with adult brain washing solution [0.5%BSA (Sigma), 0.5% TX‐100 (Sigma) in PBS], the tissues were blocked with blocking solution [3% normal goat serum (Jackson Laboratories), 3% normal donkey serum (Jackson Laboratories), 0.5% TX‐100 in PBS] for 30′. Tissues were incubated with primary antibodies overnight, washed 3 × 1 h in adult brain washing solution, incubated with secondary antibodies overnight, washed 3 × 1 h in adult brain washing solution, followed by a final wash in PBS overnight. Tissues were mounted in VectaShield (Vector Laboratories) or 50:50 VectaShield and SlowFate™ Gold (Invitrogen).
Antibodies were obtained from the Developmental Studies Hybridoma Bank (DSHB), developed under the auspices of the NICHD, and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242, as well as commercial sources. All antibodies used in this study are listed in Table2 .
Brains were fixed in 2% paraformaldehyde (PFA, Electron Microscopy Sciences) in ExpressFive™ medium for 1 h. After three or more washes (15′ each) with adult brain washing solution [0.5%BSA (Sigma), 0.5% TX‐100 (Sigma) in PBS], the tissues were blocked with blocking solution [3% normal goat serum (Jackson Laboratories), 3% normal donkey serum (Jackson Laboratories), 0.5% TX‐100 in PBS] for 30′. Tissues were incubated with primary antibodies overnight, washed 3 × 1 h in adult brain washing solution, incubated with secondary antibodies overnight, washed 3 × 1 h in adult brain washing solution, followed by a final wash in PBS overnight. Tissues were mounted in VectaShield (Vector Laboratories) or 50:50 VectaShield and SlowFate™ Gold (Invitrogen).
Antibodies were obtained from the Developmental Studies Hybridoma Bank (DSHB), developed under the auspices of the NICHD, and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242, as well as commercial sources. All antibodies used in this study are listed in Table
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