A11034

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany, China

The A11034 is a specialized laboratory instrument designed for accurate and reliable measurement. It functions as a high-precision analytical tool, providing consistent and reproducible results. The core purpose of this equipment is to facilitate precise data collection and analysis in a laboratory setting.

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Catalog A11034 (2 citations) AlexaFluor 488 conjugated-goat anti-rabbit (A11034) (2 citations) Anti-rabbit IgG labeled with Alexa Fluor 488 (A11034) (3 citations) Alexa-fluor 488 goat anti-rabbit (A11034, (3 citations) Alexa Fluor 488 anti-rabbit (A11034), (5 citations) Alexa Fluor 488 direct-labeled goat anti-rabbit (A11034) (2 citations) Alexa Fluor 488 goat anti-rabbit IgG (A11034), (14 citations)

323 protocols using a11034

Immunofluorescence Detection of MUC5AC

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After hydration in PBS, the cells were blocked for 10 min in 2.5% bovine serum albumin and 0.2% Triton X-100 diluted in PBS, followed by PBS wash and 1-h incubation at room temperature in primary antibody diluted in 0.25% bovine serum albumin and 0.05% Tween 20. Then, the cells were rinsed three times in PBS and incubated for 1 h at room temperature with the secondary antibody goat anti-rabbit Alexa Fluor 488 (1:300, A11034, Life Technologies) diluted in 0.25% bovine serum albumin and 0.05% Tween 20. After rinsing three times in PBS, the cells were mounted with VectaShield-DAPI (Vector Laboratories) to counterstain nuclear DNA.
Immunofluorescence detection of MUC5AC on bulbar conjunctival imprints was performed as described previously [34 (link)]. Briefly, primary antibody against MUC5AC and the secondary antibody Alexa Fluor 488 (1:300, A11034, Life Technologies) were diluted in 0.1% bovine serum albumin; after staining, the cells were mounted with VectaShield-DAPI (Vector Laboratories).
Immunofluorescent images were acquired with an Olympus BX51 microscope and CCD-1300 camera (VDS Vosskühler GmbH, Osnabrück, Germany). The images were analyzed with NIS Elements software (Laboratory Imaging). The percentage of positive cells was counted on six randomly captured photographs per well (two wells per condition) from at least four independent tissue donors.

Stadnikova A., Trosan P., Skalicka P., Utheim T.P, & Jirsova K. (2019). Interleukin-13 maintains the stemness of conjunctival epithelial cell cultures prepared from human limbal explants. PLoS ONE, 14(2), e0211861.

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Immunofluorescence Analysis of Neuroendocrine Markers

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NE-treated cells were fixed for 15 min using 4% paraformaldehyde and then blocked for 30 min using 5% donkey serum containing 0.3% Triton X-100. The cells were then incubated with the primary antibodies against CHGB (1 : 100; rabbit; PA5-52605, ThermoFisher Scientific) or SYP (1 : 100; rabbit; MA5-14532, ThermoFisher Scientific) for 3 and 1 h, respectively, followed by incubation with anti-rabbit Alexa Fluor® 488 Conjugate (1 : 100, A11034, ThermoFisher Scientific) secondary antibody for 1 h at room temperature. Cells were then mounted using slow-fade DAPI (S36973, Life Technologies) and examined using Axio Observer 7.
For Adrβ2 and cytokeratin expression in tumor and adjacent normal tissues, the tissues were incubated with primary antibodies against Adrβ2 (1 : 100; rabbit; PA5-14117, ThermoFisher Scientific) and cytokeratin (1 : 100; mouse; MA1-82041; ThermoFisher Scientific) for 1 h at room temperature, followed by incubation with a cocktail of anti-rabbit Alexa Fluor® 488 Conjugate (1 : 100, A11034, ThermoFisher Scientific) and anti-mouse Alexa Fluor® 546 (1 : 100, A21045, ThermoFisher Scientific) for 1 h at room temperature. Cells were then mounted using slow-fade DAPI (S36973, Life Technologies) and examined using Axio Observer 7.

Dwivedi S., Bautista M., Shrestha S., Elhasasna H., Chaphekar T., Vizeacoumar F.S, & Krishnan A. (2021). Sympathetic signaling facilitates progression of neuroendocrine prostate cancer. Cell Death Discovery, 7, 364.

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Antibody Characterization and Procurement

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Antibody information can be found in Supplementary Table S1. Antibodies against CD68 (FA-11), Rab27A and V5 were purchased from Hycult Biotechnology (HM1070), Proteintech (17817-1-AP) and Life Technologies (R96025), respectively. Antibodies to GM130 (618022) and LAMP1 (553792) were purchased from BD Biosciences Pharmingen. Antibodies against α-tubulin (DM1A, T9026), β-actin (AC-15, A5441) and FLAG (F7425) were from SIGMA. Anti-Rab7 (D95F2, 9367) and anti-Rab11 (D4F5, 5589) were obtained from Cell Signaling. Antibodies against cathepsin K (E-7, sc-48353) and GFP (B-2, sc-9996) were from Santa Cruz. Antibodies against a1, a2 and a3 were generated as described previously^{30 ,61 (link),69 (link)}. Alexa-conjugated secondary antibodies (A11034, A11029, A11081 and A21236) and colloidal gold-conjugated ones (EMGAT10 and EMGMHL5) were from Life Technologies and BBI solutions, respectively. HRP-conjugated antibodies to rabbit IgG (NA934VS), mouse IgG (NA931VS), chicken IgY (12–341) and native primary antibodies (21230) were purchased from GE healthcare (anti-rabbit IgG and mouse IgG), Millipore and Thermo Scientific.

Matsumoto N., Sekiya M., Tohyama K., Ishiyama-Matsuura E., Sun-Wada G.H., Wada Y., Futai M, & Nakanishi-Matsui M. (2018). Essential Role of the a3 Isoform of V-ATPase in Secretory Lysosome Trafficking via Rab7 Recruitment. Scientific Reports, 8, 6701.

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Immunofluorescence Microscopy of MEFs

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Formaldehyde (4%) was added to fix MEFs for 10 min and then washed with phosphate-buffered saline (PBS). To block nonspecific binding, the MEFs were incubated in a blocking solution included 10% FBS and 0.1% Triton X-100 in PBS for 15 min, followed by overnight incubation with the primary antibodies. After washing with PBS-T, the cells were treated for 90 min with the following secondary antibodies, Alexa Fluor 594-labeled goat anti-rabbit immunoglobulin G (IgG, A11037; Life Technologies, Thermo Fisher Scientific; Waltham, MA, USA), Alexa Fluor 594-labeled goat anti-mouse IgG (A11032; Life Technologies, Thermo Fisher Scientific), and Alexa Fluor 488-labeled goat anti-rabbit IgG (A11034; Life Technologies, Thermo Fisher Scientific), followed by processing using 4′,6′-diamino-2-phenylindole (DAPI), to detect cell nuclei (1:3000; 5 mg/mL stock in DMSO; Merck, Darmstadt, Germany). Cells were mounted on slides using ProLong Gold Antifade mounting medium (Molecular Probes, P36034; Thermo Fisher Scientific), and cell immunofluorescence was detected using an Olympus FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).

Wuputra K., Tsai M.H., Kato K., Yang Y.H., Pan J.B., Ku C.C., Noguchi M., Kishikawa S., Nakade K., Chen H.L., Liu C.J., Nakamura Y., Kuo K.K., Lin Y.C., Chan T.F., Wu D.C., Hou M.F., Huang S.K., Lin C.S, & Yokoyama K.K. (2021). Dimethyl sulfoxide stimulates the AhR-Jdp2 axis to control ROS accumulation in mouse embryonic fibroblasts. Cell Biology and Toxicology, 38(2), 203-222.

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Immunofluorescence Analysis of Ciliary Proteins

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Fixed cells were washed once in PBS and permeabilised in PBTX (PBS + 0.1% Triton-X100) for 10 min, washed in PBS once and then 3 times in blocking buffer (PBS + 2% BSA (Roche Diagnostics, #10735086001). Primary antibodies were diluted in blocking buffer and cells incubated for 60 min at room temperature. Cells were then washed in blocking buffer three times, and then incubated with secondary antibodies diluted in blocking buffer for 60 min at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, D9542) [1 mg/ml] diluted 1:4000 in PBS. Cells were washed thoroughly in PBS and mounted using DAKO anti-fading mounting medium (DAKO, S3023). Imaging was performed using an Olympus IX71 Personal Deltavision Deconvolution microscope or a Zeiss LSM-710 upright confocal microscope. Antibodies: acetylated alpha tubulin (mouse, Sigma-Aldrich, T6793) 1:5000, Arl13b (mouse, NeuroMab, 75–287) 1:300, gamma-tubulin (mouse, Sigma-Aldrich, T6557) 1:500, IFT88 (rabbit, Proteintech, 13967-1-AP) 1:300, CEP164 (rabbit, Sigma-Aldrich, SAB3500022) 1:200, ODF2 (rabbit, Sigma-Aldrich, HPA001874) 1:200, CP110 (rabbit, Proteintech, 12780-1-AP) 1:300, anti-mouse Alexa-594 (Life Technologies, A-11032) 1:500, anti-rabbit Alexa-488 (Life Technologies, A-11034) 1:500.

Cortés C.R., McInerney-Leo A.M., Vogel I., Rondón Galeano M.C., Leo P.J., Harris J.E., Anderson L.K., Keith P.A., Brown M.A., Ramsing M., Duncan E.L., Zankl A, & Wicking C. (2016). Mutations in human C2CD3 cause skeletal dysplasia and provide new insights into phenotypic and cellular consequences of altered C2CD3 function. Scientific Reports, 6, 24083.

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Oligodendrocyte Differentiation Assay

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Pure OPC (POPC) cultures were generated from male and female WT P5-9 mice as previously described (45 (link)). At day 9 in culture, PDGFαα and NeuroTrophin 3 (NT3) were withdrawn to allow oligodendrocyte differentiation and cells were stimulated with 5% Treg-CM, 5% CCN3-depleted Treg-CM, 5% IgG isotype-depleted Treg-CM, or controls for up to 3 d. Purity was assessed in random cultures at 3–5 d after setup and was over 93%.
POPCs were fixed directly in 4% PFA (pH 7.4) (Sigma) for 15 min at RT. POPCs were blocked in 10% NGS (Vector Laboratories s-1000) with 0.1% Triton X-100 in PBS for 1 h. Cells were then incubated with primary antibodies for Olig2 (1:400 Millipore AB9610) and MBP (1:1,000 Millipore MAB386) in 2% NGS with 0.01% Triton X-100 in PBS overnight at 4 °C. Afterward, OPCs were incubated with secondary antibodies (1:1,000, goat anti-rabbit 488, A11034 and 1:1,000, goat anti-rat 594, A11007, both Life Technologies) for 1 h at RT. Cells were stained with DAPI for 5 min at RT. Immunofluorescence was detected using an EVOS FL microscope at 10× magnification and n = 5 wells per condition.

de la Vega Gallardo N., Penalva R., Dittmer M., Naughton M., Falconer J., Moffat J., de la Fuente A.G., Hombrebueno J.R., Lin Z., Perbal B., Ingram R.J., Evergren E, & Fitzgerald D.C. (2020). Dynamic CCN3 expression in the murine CNS does not confer essential roles in myelination or remyelination. Proceedings of the National Academy of Sciences of the United States of America, 117(30), 18018-18028.

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Immunofluorescence Staining Protocol for Vimentin and Cytokeratin 19

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Firstly, the prepared cell slides were washed with PBS. The slides were fixed with 4% paraformaldehyde for 30 minutes at room temperature. After washing with PBS 3 times for 5 minutes, the slide was incubated with 0.1% Triton X-100 solution (sc281181; Santa Cruz, CA, USA) for 10 minutes at room temperature. After that, 1% BSA solution was added to each well and blocked at 37°C for 90 minutes. The slides were incubated with primary antibodies including anti-vimentin (1: 100; 10366-1-AP; Proteintech, USA) and anti-cytokeratin 19 (1: 100; 10712-1-AP; Proteintech, USA) overnight at 4°C in a refrigerator. Next, the cells were incubated with goat anti-rabbit IgG (H+L) 488 fluorescent secondary antibody (1: 800; A11034; Life Technologies, USA) at 37°C for 1 hour in the dark. The nucleus was stained with DAPI (D9452; SIGMA, Canada) for 5 minutes at room temperature. Finally, the slides were observed under a fluorescence microscope.

Xu F., Shen X., Sun C., Xu X., Wang W, & Zheng J. (2020). The Effect of Mitomycin C on Reducing Endometrial Fibrosis for Intrauterine Adhesion. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e920670-1-e920670-9.

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Immunoblot and Immunofluorescence Protocols for AQP4 Detection

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For the immunoblot and for immunofluorescence experiments the following primary antibodies were used: rabbit anti-human AQP4ex [6 (link)] used at a concentration of 0.3 μg/mL, custom rabbit anti-AQP4 were generated using the c-terminus sequence as previously described [10 (link)] (GenScript Biotech, Piscataway, NJ, USA) used at a concentration of 0.13 μg/mL for immunoblot and at 0.4 μg/mL for immunofluorescence. The secondary antibody used for immunoblotting experiments was anti-rabbit IgG-HRP (Bio-Rad Cat# 172–1019, RRID:AB_11125143) and for immunofluorescence AlexaFluor 488 anti-rabbit was used at a concentration of 1 μg/mL (Life Technologies, Thermo Fisher Scientific Cat# A-11034, RRID:AB_2576217).

Valente O., Messina R., Ingravallo G., Bellitti E., Zimatore D.S., de Gennaro L., Abbrescia P., Pati R., Palazzo C., Nicchia G.P., Trojano M., Signorelli F, & Frigeri A. (2022). Alteration of the translational readthrough isoform AQP4ex induces redistribution and downregulation of AQP4 in human glioblastoma. Cellular and Molecular Life Sciences, 79(3), 140.

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Immunocytochemical Analysis of Stem Cell Markers

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Cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.25% Triton X-100 and blocked with 5% FCS in PBS for 1 h. The fixed cells were incubated overnight at 4 °C in PBS + 1% FCS with antibodies mouse anti-OCT4 (1:500, ab18976, Abcam, Cambridge, MA), rabbit anti-SOX2 (1:500, MA516399, ThermoFisher), rabbit anti-TUJ1 (1:500, MAB1195, R&D system, Minneapolis, MN), rabbit anti-OLIG2 (1:500, NBP128667, Novus), rabbit anti-HB9 (1:500, ABN174, Millipore-Sigma, Temecula, CA), rabbit anti-CHAT (1:500, AB144P, Abcam, Cambridge, MA), and rabbit anti-cleaved caspase-3 (CC3, 1:500, 9669S, Cell Signaling Technology) followed by incubation with secondary antibodies: FITC-conjugated anti-mouse IgG (1:1000, A21202, Life Technologies), FITC-conjugated anti-rabbit IgG (1:1000, A11034, Life Technologies), Cy3-conjugated anti-mouse IgG (1:1000, A11003, Life Technologies), and Cy3-conjugated anti-rabbit IgG (1:1000, A11035, Life Technologies). The treated cells were covered with Slowfade antifade with DAPI (Life Technologies) for nuclear staining and covered with a glass coverslip. Images were captured with a fluorescence microscope (DM5000B, Leica, Wetzlar, Germmany).

Kim D., Kim S., Sung A., Patel N., Wong N., Conboy M.J, & Conboy I.M. (2022). Autologous treatment for ALS with implication for broad neuroprotection. Translational Neurodegeneration, 11, 16.

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Immunofluorescence Staining of Locust Brain Tissues

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After the dissected brain tissues were fixed in 4% formaldehyde at 4 °C overnight, they were washed with 0.1 M PBS twice for 15 min each (pH 7.4). Thick sections of the brains (30 μm) were cut with a vibrating blade microtome (VT 1200 S, Leica, Wetzlar, Germany), washed in 0.1 M PBS for 15 min, and then incubated in 0.1 M PBS containing 5% normal goat serum (NGS, Boster, China) for 1 h at room temperature. The primary anti-Dop1 and anti-AC2 (custom made, see “Protein preparation and Western blot analysis” section of Methods for details) was diluted at 1:300 in 0.1 M PBS containing 2% NGS. Incubation with primary antibodies lasted for 48 h. The tissues were washed with 0.1 M PBS three times for 15 min each and subsequently incubated with the mixture of two secondary antibodies, Goat anti-rabbit antibody Alexa fluor 488 (1: 500, A11034, Life Technology) and Goat anti-mouse antibody Alexa fluor 546 (1: 500, A11030, Life Technology), for 1 h at room temperature. After washing three times, the tissues were mounted in anti-fade fluorescence mounting medium. The negative serum of Dop1 and AC2 from rabbit and mouse were used as the negative control. The nucleus of locust brain is labeled by Hoechst33342 (Life Technology) to indicate the brain structure. The fluorescence was detected using a Zeiss LSM 710 confocal microscope (Zeiss, Oberkochen, Germany).

Guo X., Ma Z., Du B., Li T., Li W., Xu L., He J, & Kang L. (2018). Dop1 enhances conspecific olfactory attraction by inhibiting miR-9a maturation in locusts. Nature Communications, 9, 1193.

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