One to three-day-old flies were used when mapping the connectivity rate between input neurons and α/βp Kenyon cells. Brains were dissected in saline, treated for 1 min with 2 mg/ml collagenase (Sigma-Aldrich) and mounted on a piece of Sylgard placed at the bottom of a Petri dish. The imaging protocol is the same as described above but the photo-labeling protocol is different. Each of the input neurons was photo-labeled using a single plane centered on either its soma or its projection and by scanning the plane three to five times. Each pixel was scanned eight times with a pixel size of 0.019 μm and a pixel dwell time of 4 μs. A fire-polished borosilicate glass pipette (0.5 mm I.D., 1.0 mm O.D., 10 cm length; Sutter Instruments) was pulled using the P-2000 micropipette puller (Sutter Instruments) and backfilled with Texas Red dye (lysine-fixable 3000 MW; Life Technologies) dissolved in saline. The tip of the pipette was positioned next to the cell body of a randomly chosen α/βp Kenyon cell under the two-photon microscope. The dye was electroporated into the cell body using three to five 1–5 ms pulses of 20–50 V. The dye was allowed to diffuse within the Kenyon cell for 5 min before the brain was imaged.
Texas red dye
Manufactured by Thermo Fisher Scientific
Texas Red dye is a fluorescent dye used in various laboratory applications. It has an excitation maximum at 596 nm and an emission maximum at 613 nm, making it suitable for detection and labeling purposes in fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Collagenase, by Merck Group (2 mentions)
P 2000 micropipette puller, by Sutter Instruments (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Alkaline phosphatase, by Promega (1 mentions)
Cellmask orange, by Thermo Fisher Scientific (1 mentions)
Nhs fitc, by Thermo Fisher Scientific (1 mentions)
Anti gst, by Cell Signaling Technology (1 mentions)
Oti1e4, by OriGene (1 mentions)
Ma5 15256, by Thermo Fisher Scientific (1 mentions)
Anti penta his, by Qiagen (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Sir tubulin, by Spirochrome (1 mentions)
P38 sc 7149, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Sb203580, by Merck Group (1 mentions)
Middlebrook 7h10 agar, by BD (1 mentions)
Middlebrook 7h9 broth medium, by BD (1 mentions)
8 protocols using texas red dye
1
Mapping Connectivity between Neurons and Kenyon Cells
2
Mapping Connectivity between Neurons and Kenyon Cells
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One to three-day-old flies were used when mapping the connectivity rate between input neurons and α/βp Kenyon cells. Brains were dissected in saline, treated for 1 min with 2 mg/ml collagenase (Sigma-Aldrich) and mounted on a piece of Sylgard placed at the bottom of a Petri dish. The imaging protocol is the same as described above but the photo-labeling protocol is different. Each of the input neurons was photo-labeled using a single plane centered on either its soma or its projection and by scanning the plane three to five times. Each pixel was scanned eight times with a pixel size of 0.019 μm and a pixel dwell time of 4 μs. A fire-polished borosilicate glass pipette (0.5 mm I.D., 1.0 mm O.D., 10 cm length; Sutter Instruments) was pulled using the P-2000 micropipette puller (Sutter Instruments) and backfilled with Texas Red dye (lysine-fixable 3000 MW; Life Technologies) dissolved in saline. The tip of the pipette was positioned next to the cell body of a randomly chosen α/βp Kenyon cell under the two-photon microscope. The dye was electroporated into the cell body using three to five 1–5 ms pulses of 20–50 V. The dye was allowed to diffuse within the Kenyon cell for 5 min before the brain was imaged.
3
Live-Cell Imaging of Yeast Mitosis
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Video microscopy was performed as described, except that cells were not fixed and were imaged in low-fluorescence SC medium. G1-arrested cells were washed three times with SC medium to remove α-factor, adhered to concanavalin A–coated coverslips, and imaged for up to 3 h. For anaphase experiments (Supplemental Figure S3), images were collected every 5 min. For metaphase arrest experiments (Figure 5, E and F ), images were collected every 10 min. for experiments in which wild-type and plasmid-containing cells were imaged together (Supplemental Figure S3), wild-type cells walls were labeled with Texas red dye (Life Technologies). Cells were washed twice with 1× phosphate-buffered saline, resuspended in 0.1 M NaHCO3, pH 8.3, incubated for 5 min with 10 mg/ml DMSO stocks of Texas red at a final concentration of 10 μg/ml, quenched by washing three times with 0.1 M NaHCO3, pH 8.3, plus 200 μM lysine, and washed twice with SC medium. Plasmid-containing cells were mock labeled by incubating with DMSO alone and washed as described.
4
Campylobacter jejuni Microcrystal Production
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Microcrystal-forming protein derived from Campylobacter jejuni was expressed, purified, and crystallized as described previously (PDB entry 5w17)(30 (link), 32 ). Microcrystals were crosslinked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or glyoxal during trace-labeling with Texas Red dye (ThermoFisher) as described previously (33 (link)). See Supplementary Material (Porous Crystal Production, Crystal Fluorophore Labeling) for additional details regarding microcrystal growth, crosslinking, and labeling.
5
Antibody Sources and Reagents for Protein Analysis
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The primary antibody against SNX9 (OTI1E4) was purchased from Origene, anti-EGFR (PA1-1110) and anti-GFP (MA5-15256) were from Thermo Scientific, antipenta-His (#34660) from Qiagen, anti-GST (#2622) from Cell Signaling, and anti-DsRed (sc-101526) from Santa Cruz. Anti-IncA was a gift from G. Zhong, University of Texas Health Science Center at San Antonio, San Antonio, TX (57 (link)), and anti-DnaK was obtained from S. Birkelund, Aalborg University, Aalborg, Denmark (58 (link)). Antibodies against Cpn0677 and Cpn0678 were generated in our laboratory, as was anti-CPn0147. Mouse anti-Cya, rabbit anti-CRP, and rabbit anti-IpaD antibodies were generously donated by N. Guiso, A. Ullmann, and C. Parsot, Institut Pasteur, Paris, France, respectively. Secondary anti-rabbit and anti-mouse antibodies coupled to Alexa488 or Alexa594 were purchased from Thermo Scientific, and those coupled to alkaline phosphatase were sourced from Promega. CellMask (orange) and Rhodamine-Phalloidin were purchased from Thermo Scientific, and SiR-Tubulin from Spirochrome. All lipids used in this study were obtained from Avanti Lipids, and Texas red dye, NHS-FITC and DyLight650NHS from Thermo Scientific. Nocodazole and Cytochalasin D were purchased from Merck.
6
BODIPY-MC Labeling of OVA
7
Investigating TRAF6 Signaling Pathways
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Middlebrook 7H10 agar and Middlebrook 7H9 broth medium were purchased from BD Difco Laboratories (Sparks, MD). Texas Red dye was obtained from Invitrogen (Carlsbad, CA). Antibodies against NF-κB p65 (sc-109), Lamin B (sc-6216), JNK (sc-571) and p38 (sc-7149) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal (ab33915) and polyclonal (sc-7221) antibodies against TRAF6 were from Abcam and Santa Cruz Biotechnology, respectively. Antibodies against ERK1/2 (#4695), phosphorylated ERK1/2 (#4370), phosphorylated JNK (#4668) and phosphorylated p38 (#9215) were obtained from Cell Signaling Technology (Beverly, MA). The β-actin (A1978) antibody was obtained from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG and ProLong Gold antifade reagent were from Invitrogen. Inhibitors of IKKα/β (BMS345541), JNK (SP600125) and p38 (SB203580) were obtained from Merck (Darmstadt, Germany).
8
Thermal Stability Assessment of T7 RNA Polymerase
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The relative thermal stability of each T7 RNA polymerase was assessed by incubating 0.5 mg/ml enzyme in PBS buffer with TexasRed dye (Invitrogen). Enzyme/dye mixtures were equilibrated at 37°C for 10 min and heated at a rate of 0.07°C/s to 97°C using a LightCycler 96 thermocycler, while fluorescence was monitored (Excitation 577 nm / Emission 620 nm). The first derivatives of the change in fluorescence as a function of time were used to approximate the relative Tm. Data were analyzed using Roche thermocycler software.
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