Alexa fluor 488 goat anti rabbit

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Alexa Fluor 488 goat anti-rabbit is a fluorescently labeled secondary antibody used for detection and visualization in various immunological and cell biology applications. It is produced by conjugating the Alexa Fluor 488 dye to goat-derived antibodies that specifically bind to rabbit primary antibodies.

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Goat anti-rabbit IgG (H + L) cross-adsorbed Alexa Fluor 488 Goat anti-rabbit IgG (H+L) Alexa Fluor 488 conjugate Alexa Fluor 488-or Alexa Fluor 568-labeled goat anti-rabbit antibodies Alexa Fluor 488®-conjugated goat anti-rabbit IgG (H + L) Alexa Fluor 488 F(ab′)2 fragment of goat anti-rabbit IgG antibody Alexa Fluor 488-labeled goat anti-rabbit Alexa Fluor R 488 Goat Anti–rabbit Goat Alexa-fluor 488-conjugated anti-rabbit IgG antibodies Alexa Fluor 488-coupled goat anti-rabbit IgG antibody Alexa Fluor 488-conjugated goat anti-rabbit

709 protocols using alexa fluor 488 goat anti rabbit

Immunofluorescence Staining of Cells and Tissues

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Sc were cultured and fixed in 2% paraformaldehyde. The cells were permeabilized using 0.1% triton X-100 for 2 min followed by blocking with 3% BSA for 30 min. Primary antibody (Abcam, Cat. no. ab12132) was used at a dilution of 1:100 (overnight incubation, 4 °C). Secondary antibody alexafluor 488- goat anti-rabbit (Life Technologies, Cat. no. A-11008) was used at a dilution of 1:500 (45 min incubation, RT), Nuclei were stained using Hoechst 3342. The coverslips were mounted on glass slides using prolong gold Antifade reagent (Invitrogen). Cell imaging was done using Ri-2 Epi-flourescence microscope (Eclipse TiNikon).
Testes were fixed in 4% paraformaldehyde followed by paraffin block preparation and tissue sectioning as described above (Histological analysis). The tissue sections were permeabilized in 0.5% triton X-100 for 20 min followed by blocking with 2% normal goat serum for 30 min. Primary antibody (Abcam, Cat. no. ab12132) was used at a dilution of 1:100 (overnight incubation, 4 °C). Secondary antibody alexafluor 488-goat anti-rabbit (Life Technologies, Cat. no. A-11008) was used at a dilution of 1:500 (4-h incubation, RT), Nuclei were stained using Hoechst 3342. Coverslips were mounted over the tissue sections using prolong gold Antifade reagent (Invitrogen). Tissue imaging was performed using Ri-2 Epi-flourescence microscope (Eclipse Ti Nikon).

Mandal K., Bader S.L., Kumar P., Malakar D., Campbell D.S., Pradhan B.S., Sarkar R.K., Wadhwa N., Sensharma S., Jain V., Moritz R.L, & Majumdar S.S. (2017). An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation. DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes, 24(2), 143-157.

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Fluorescent Imaging of NOX4, IL-1β, and Mitochondrial Activity

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Fluorescence staining of lung tissue and BEAS-2B cells was performed. Briefly, primary rabbit monoclonal antibody against NOX4 (ab133303, Abcam) used for incubation overnight at 4°C, followed by incubation with AlexaFluor488 goat anti-rabbit (A11001, Life Technologies, Waltham, MA, USA) for 2 h. BEAS-2B cells were stained with primary antibody against IL-1β (12703, CST) overnight at 4°C and then with AlexaFluor488 goat anti-rabbit (A11001, Life Technologies) for 2 h. Mitochondrial membrane potential (MMP) fluorescence staining was performed using tetramethylrhodamineethylester (TMRE) (113852, Abcam) at a final concentration of 50 nM at 37°C for 30 min. The mtROS fluorescence staining was performed using MitoSOX (M36008, mitoSOX mitochondrial superoxide Indicator, Thermo Fisher) at a final concentration of 5 μM at 37°C for 10 min. All data were collected by Cytation ™ 5 (BioTek Instruments).

Wang Z., Song Y., Jiang J., Piao Y., Li L., Bai Q., Xu C., Liu H., Li L., Piao H, & Yan G. (2022). MicroRNA-182-5p Attenuates Asthmatic Airway Inflammation by Targeting NOX4. Frontiers in Immunology, 13, 853848.

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Immunohistochemical and Biochemical Analysis of BRI2 Amyloidosis

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For immunohistochemical and biochemical studies, we use antibodies against the ABri and ADan amyloid peptides which also recognize the C-terminus of the immature forms of mutant BRI₂ (Vidal et al., 2009 (link)). We also used abs against the N-terminus of BRI₂: ab14307 (Abcam) and 11–26 (Akiyama et al., 2004 (link)). Abs against c-Myc (Santa Cruz) and against beta-actin (AC-15, Sigma) were also used. Secondary abs used for Western blot ECL detection were donkey anti-rabbit IgG, HRP (NA934, GE Healthcare), anti-mouse IgG, HRP (NA931, GE Healthcare), and anti-chicken IgY, HRP (A9046, Sigma-Aldrich). For confocal studies, BRI₂ proteins were detected with anti-c-Myc and secondary Alexa Fluor 594 goat anti-mouse (Invitrogen). Calnexin was detected with anti-Calnexin (Abcam) and secondary Alexa Fluor 488 goat anti-rabbit (Invitrogen). GM130 was detected with anti-GM130 (Abcam) and secondary Alexa Fluor 488 goat anti-rabbit (Invitrogen).

Garringer H.J., Sammeta N., Oblak A., Ghetti B, & Vidal R. (2016). Amyloid and intracellular accumulation of BRI2. Neurobiology of aging, 52, 90-97.

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Immunohistochemical Analysis of Muscle Proteins

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Immunohistochemistry of Drosophila larval body wall muscles and human muscle biopsy tissues were performed as previously described [9 (link)]. Drosophila larval body wall muscles were stained with affinity purified anti-CncC antibodies (1:100; gift from H. Deng and T. Kerppola) [24 (link)], anti-p62/Ref(2)P (1:3,000, kind gift of G. Juhász) [55 (link)] that was detected with Alexa Fluor 488 goat anti-rabbit (1:400 dilution; Invitrogen). Filamentous actin was detected with Texas Red Phalloidin (1:400, Invitrogen). Human muscle biopsy cryosections were obtained from the Iowa Wellstone Muscular Dystrophy Cooperative Research Center and stained according to published procedures [9 (link)] with human p62/SQSTM1 (1:3,000, Sigma) and Nrf2 (1:300, Santa Cruz Biotech), followed by Alexa Fluor 488 goat anti-rabbit (1:400, Invitrogen). Filamentous actin was detected with Texas Red Phalloidin (1:400, Invitrogen).

Dialynas G., Shrestha O.K., Ponce J.M., Zwerger M., Thiemann D.A., Young G.H., Moore S.A., Yu L., Lammerding J, & Wallrath L.L. (2015). Myopathic Lamin Mutations Cause Reductive Stress and Activate the Nrf2/Keap-1 Pathway. PLoS Genetics, 11(5), e1005231.

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Quantitative Analysis of Nuclear STAT3 Localization

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Cells were cultured for 48 hours on glass coverslips in 6-well plate and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, followed by permeabilization with 0.1% Triton X-100 for 5 min at room temperature, incubation with primary antibody for one hour, wash with 1% Triton X-100, and incubation with Alexa Fluor-conjugated secondary antibodies for one hour in the dark. The coverslips were stained with Hoechst 33342 (H3570, Invitrogen, 1:3000) before being mounted with VECTASHIELD mounting medium (H-1000, Vector laboratories). Images were captured by Leica TCS-NT 4D confocal microscope. Z stacks were collected with a spacing of 1 μm. Antibody and dilutions used in the studies: anti-Flag (14793S, Cell signaling, 1:100), anti-Lamin B1 (68591S, Cell signaling, 1:100), anti-STAT3 (MA1–13042, Thermo Fisher Scientific, 1:100). Alexa Fluor 488 Goat anti-Rabbit (A11008, Life technologies, 1:5000), Alexa Fluor 594 Goat anti-Mouse (A11032, Life technologies, 1:5000). The fluorescence-intensity profile along the Z-axis from confocal Z-stacks were shown. The fluorescence intensity of nuclear localized STAT3 was quantified using the confocal software to define the selected ROI (Region of Interest) area based on nuclear DAPI signal. More than 200 cells were quantified in at least three independent experiments.

Niu J., Sun Y., Chen B., Zheng B., Jarugumilli G.K., Walker S.R., Hata A.N., Mino-Kenudson M., Frank D.A, & Wu X. (2019). Fatty acids and cancer-amplified ZDHHC19 promote STAT3 activation through S-Palmitoylation. Nature, 573(7772), 139-143.

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Antibody Characterization for IF and IB

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The following antibodies were used as primary antibodies for immunofluorescence microscopy: SGO1 (SAB1405371, Sigma Aldrich), GFP (ab290, Abcam) and CENPA (07–574, Millipore; and ab13939, Abcam). For immunoblotting, the following primary antibodies were used: SA1 (ab4457, Abcam), SA2 (A300-158a, Bethyl Laboratories), SMC1 (A300-055A, Bethyl Laboratories), SCC1 (05-908, Millipore), WAPL (A-7, sc-365189, Santa Cruz), Sororin (ab192237, Abcam), HSP90 (sc-13119(F-8), Santa Cruz) and α-tubulin (T5168, Sigma Aldrich). All primary antibodies were used at a 1:1000 dilution with the exception of HSP90 and α-tubulin (1:10000). For coimmunoprecipitation, we used 4.5 μg of SMC1 (A300-055A, Bethyl Laboratories) or IgG (2729 S, Cell Signaling) per sample. Secondary antibodies were used at a 1:1000 dilution. For immunofluorescence microscopy we used: Alexa Fluor 488 goat anti-mouse (A-11001, Life Technology), Alexa Fluor 568 goat anti-mouse (A-11004, Life Technology), Alexa Fluor 488 goat anti-rabbit (A-11008, Life Technology) and Alexa Fluor 568 goat anti-rabbit (A-11011, Life Technology). For western blots, we used the following secondary antibodies: anti-goat-PO (P0449, DAKO), anti-rabbit-PO (P0448, DAKO) and anti-mouse-PO (P0447, DAKO).

García-Nieto A., Patel A., Li Y., Oldenkamp R., Feletto L., Graham J.J., Willems L., Muir K.W., Panne D, & Rowland B.D. (2023). Structural basis of centromeric cohesion protection. Nature Structural & Molecular Biology, 30(6), 853-859.

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Evaluating Ubiquitin Linkages in Plasmodium

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P. falciparum 3D7 isolate was maintained under standard conditions (Moll, 2013 ; Radfar et al., 2009 (link)). Synchronized ring-stage P. falciparum parasites (10 h post-reinvasion) at 10% parasitemia, 1% hematocrit were treated with either takinib (0.001–100 μM), compound 5 (30 μM), HS220 (30 μM), or ≤ 0.1% DMSO. After 24 hrs, cultures were harvested, treated with 0.03% saponin lysis buffer and then lysed by sonication. Lysates were analyzed by Western blot with K63-linkage specific anti-ubiquitin (Abcam ab179434) and Alexa Fluor 488 goat anti-rabbit (Life Technologies A11008). K48-linkage specific anti-ubiquitin (Abcam ab140601) was detected as a control using secondary Alexa Fluor 647 goat anti-rabbit IgG antibody (Life Technologies, A32733). Anti-actin (Abcam ab3280) was used as a loading control.

Raphemot R., Eubanks A.L., Toro-Moreno M., Geiger R.A., Hughes P.F., Lu K.Y., Haystead T.A, & Derbyshire E.R. (2018). Plasmodium PK9 Inhibitors Promote Growth of Liver Stage Parasites. Cell chemical biology, 26(3), 411-419.e7.

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Arabidopsis Seedling Immunofluorescence Assay

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Nine-day-old Arabidopsis seedlings were fixed in 4% PFA and processed as described previously on nuclei from squashed root tip (Batzenschlager et al., 2015 (link)). The primary anti-γ-H2AX antibody (diluted at 1/500) produced by Davids Biotechnology (Regensburg, Germany; Friesner et al., 2005 (link)), the rat anti-RAD51 antibody (diluted at 1/500) (Kerzendorfer et al., 2006 (link)) and when needed the monoclonal antibody directed against GFP (diluted at 1/500) (Invitrogen, Thermo Fisher Scientific) were incubated overnight at 4°C. Signals were revealed accordingly with the following secondary antibodies: Alexa fluor-488 goat anti-rabbit (diluted at 1/200), the Alexa fluor-488 goat anti-mouse (diluted at 1/200), the Alexa fluor-568 goat anti-rat (diluted at 1/300) and the Cy5 goat anti-rabbit (diluted at 1/300) (Life Technologies, Thermo Fisher Scientific). Root tips were mounted in antifade Vectashield (Vector Laboratories), with DAPI (2 μg/ml).

Singh G., Batzenschlager M., Tomkova D., Herzog E., Hoffmann E., Houlné G., Schmit A.C., Berr A, & Chabouté M.E. (2022). GIP1 and GIP2 Contribute to the Maintenance of Genome Stability at the Nuclear Periphery. Frontiers in Plant Science, 12, 804928.

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Immunofluorescence and Western Blot Antibody Dilutions

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The following antibodies were used for IF and diluted in TBS supplemented with 0.1% Tween20 and 2% BSA: CyclinA2 (1:400; #sc-751; Santa Cruz), CyclinA2 (1:400; #4656; Cell Signaling), PLK1 (1:400; ab14210; Abcam), pTCTP (1:400; #5251; Cell Signaling), pH2AX (1:800; #2577; Cell Signaling), pCyclinB1 (1:400; #ab55184; Abcam), pLaminA/C (1:400; #2026; Cell Signaling), Alexa Fluor 488-Goat anti-Rabbit (1:2000; #A11008 Life Technologies) and Alexa Fluor 555-Goat anti-Mouse (1: 2000; #A21422 Life Technologies). The following antibodies were used for WB and diluted in TBS supplemented with 0.1% Tween20 and 5% milk powder: CDC6 (1:400; #sc-9964 Santa Cruz), CDT1 (1:400; #sc-28262 Santa Cruz), GAPDH (1:20000; #G8795; Sigma), anti-Rabbit HRP (1:2000; #ab6721; Abcam) and anti-Mouse HRP (1:2000; #ab97023; Abcam).

Lemmens B., Hegarat N., Akopyan K., Sala-Gaston J., Bartek J., Hochegger H, & Lindqvist A. (2018). DNA Replication Determines Timing of Mitosis by Restricting CDK1 and PLK1 Activation. Molecular Cell, 71(1), 117-128.e3.

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Immunostaining of BMAL1 in Brain

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Briefly, animals were perfused transcardially using phosphate buffer containing 4% paraformaldehyde and 0.2% picric acid. Brain samples were stained for BMAL1 following previous reported methods with slight modifications [18 (link)]. Briefly, rabbit anti-BMAL1 (Abcam, Cambridge, UK; 1:1000) was used as the primary antibody and Alexa fluor 488 goat anti-rabbit (Life Technologies, Carlsbad, CA, USA; 1:500) as the secondary antibody.

Nakata M., Kumari P., Kita R., Katsui N., Takeuchi Y., Kawaguchi T., Yamazaki T., Zhang B., Shimba S, & Yada T. (2021). Circadian Clock Component BMAL1 in the Paraventricular Nucleus Regulates Glucose Metabolism. Nutrients, 13(12), 4487.

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