A11041

Manufactured by Thermo Fisher Scientific

Sourced in United States

The A11041 is a laboratory instrument designed for spectrophotometric analysis. It is capable of accurately measuring the absorbance of light by samples across a range of wavelengths, facilitating various analytical procedures in scientific research and testing applications.

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Lab products found in correlation

13 protocols using a11041

Immunofluorescence and Western Blot Protocols

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The following antibodies and dilutions were used in this study for immunofluorescence experiments: mouse anti-myc (1:200, #SC-40; Bio Connect), chicken anti–β-galactosidase (1:2,500, BGL-1040; Aveslab), mouse anti-Rab3 (1:200, 610379; BD Biosciences), rabbit anti-Arl8A (1:200, 17060-1-AP; Proteintech), rabbit anti-Arl8B (1:200, 13049-1-AP; Proteintech), rabbit anti-TRIM46 serum (1:500, described before [van Beuningen et al., 2015 (link)]), goat anti-chicken Alexa Fluor 405 (1:400, ab175675; Abcam), goat anti-rabbit Alexa Fluor 405 (1:400, A31556; Thermo Fisher Scientific), goat anti-mouse Alexa Fluor 488 (1:400, A11029; Thermo Fisher Scientific), goat anti-rabbit Alexa Fluor 488 (1:400, A11034; Thermo Fisher Scientific), goat anti-mouse Alexa Fluor 568 (1:400, A11031; Thermo Fisher Scientific), goat anti-chicken Alexa Fluor 568 (1:400, A11041, Thermo Fisher Scientific), and goat anti-mouse Alexa Fluor 647 (1:400, A21236; Thermo Fisher Scientific). For live-imaging analysis, NF-CF555 (Farías et al., 2016 (link)) was used. For Western blot, the following antibodies were used: mouse anti-myc (1:200, SC-40; Bio Connect), rabbit anti-GFP (1:10,000, ab290; Abcam), goat anti-mouse IRDye800CW (1:15,000, 926-32210; LI-COR), and goat-anti-rabbit IRDye680LT (1:20,000, 926-68021; LI-COR). Reagents used in this study are rapalog (AP21967, 635056; TaKaRa), KN-93, and KN-92.

Hummel J.J, & Hoogenraad C.C. (2021). Specific KIF1A–adaptor interactions control selective cargo recognition. The Journal of Cell Biology, 220(10), e202105011.

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Cryo-Preserved Retinal Tissue Sectioning

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Whole eyes were cryo-preserved by immersion in 30% sucrose overnight at 4 °C, followed by embedding in optimal cutting temperature compound (OCT) (Sakura Finetek). Eyes were then frozen on dry ice and 13 μm sections collected through the dorsal–ventral/superior–inferior axis of the retina onto super frost plus slides using a OTF5000 cryostat (Bright instruments). Sections were washed in PBS and blocked in 5% normal goat serum (NGS) (G2023; Sigma-Aldrich), 2% BSA, and 0.3% Triton X-100 in PBS for 60 min at room temperature. Sections were then incubated in primary antibodies against RBPMS (1832; 1:500; PhosphoSolutions), Prox1 (925202; 1:500; BioLegend), Calretinin (ab702; 1:500; Abcam), Calbindin (ab11426; 1:500; Abcam), PKC-α (sc-8393; 1:500; Santa Cruz Biotechnology) or Vimentin (ab5733; 1:500; Millipore). Afterwards, the tissue was washed in PBS then incubated with secondary antibodies anti-guinea pig AF 555 (A21435; 1:1000; Thermo Fisher Scientific), anti-chicken AF 568 (A11041; 1:1000; Thermo Fisher Scientific), anti-rabbit AF 647 (A32733; 1:1000; Thermo Fisher Scientific) or anti-mouse AF 555 (A21424; 1:1000; Thermo Fisher Scientific) with DAPI (D1306; 1:8000; Thermo Fisher Scientific) in the above-mentioned blocking detergent for 2 h at room temperature. The retinal sections were washed in PBS and then mounted using FluorSave^TM reagent.

Nieuwenhuis B., Laperrousaz E., Tribble J.R., Verhaagen J., Fawcett J.W., Martin K.R., Williams P.A, & Osborne A. (2023). Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters. Gene Therapy, 30(6), 503-519.

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Quantification of Astrocyte Markers in Tissue

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The sections were blocked in goat serum before overnight incubation with primary antibodies at 4° C. After washing, fluorescent secondary antibody incubation was undertaken for two hours at room temperature. Primary antibodies were anti-aquaporin-4 (AQP4) (EMD Millipore, AB3594; 1:500) and anti-glial fibrillary acidic protein (GFAP) (Thermofisher, PA1-10004; 1:400). Secondary antibodies were goat anti-rabbit AF568 (Thermofisher, A11011; 1:500) and goat anti-chicken AF568 (Thermofisher, A11041; 1:500), respectively. For both hemispheres, we acquired 10 µm z-stacks from six mice using a Nikon Instruments C2+ confocal laser-scanning microscope equipped with a 40× 1.3 NA oil immersion objective. The excitation sources were 405, 488, and 565 nm solid-state diode laser lines. Maximum intensity projections were analyzed in ImageJ software.

von Holstein-Rathlou S., Petersen N.C, & Nedergaard M. (2017). Voluntary running enhances glymphatic influx in awake behaving, young mice. Neuroscience letters, 662, 253-258.

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Immunofluorescence Detection of BioID2 Fusion Proteins

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Cells grown on 1.5 mm glass coverslips were fixed with 3% (w/v) paraformaldehyde/phosphate-buffered saline for 10 min and permeabilized with 0.4% (w/v) Triton X −100/PBS for 15 min. To detect BioID2 fusion proteins, chicken anti-BioID2 (1:5000; BID2-CP-100; BioFront Technologies) was used (18 (link)). The anti-BioID2 antibody was detected using Alexa Fluor 568–conjugated goat anti-chicken (1:1000; A11041; Thermo Fisher Scientific). Alexa Fluor 488–conjugated streptavidin (1:1000; S32354; Thermo Fisher Scientific) was used to detect biotinylated proteins. DNA was detected with Hoechst dye 33342. Coverslips were mounted using 10% (wt/vol) Mowiol 4 to 88 (Polysciences). Epifluorescence images were obtained using a Nikon Eclipse NiE microscope (40 × /0.75 Plan Apo Nikon objective) with a charge-coupled device camera (CoolSnap HQ; Photometrics) linked to a workstation running NIS-Elements software (Nikon). All images were processed in Adobe Photoshop CC 2023 (https://www.techspot.com/downloads/6043-adobe-creative-cloud-photoshop.html) (Adobe) for cropping and brightness/contrast adjustment when applicable.

Torres H.M., Fang F., May D.G., Bosshardt P., Hinojosa L., Roux K.J, & Tao J. (2023). Comprehensive analysis of the proximity-dependent nuclear interactome for the oncoprotein NOTCH1 in live cells. The Journal of Biological Chemistry, 300(1), 105522.

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Autaptic Hippocampal Neuron Immunostaining

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Autaptic hippocampal neurons were fixed at DIV14 in 2% paraformaldehyde (PFA) in culture medium for 10 min and subsequently in 4% PFA for 10 min. Cells were then washed with phosphate-buffered saline (PBS), permeabilized by 0.5% Triton X-100 for 5 min, and blocked with 4% normal goat serum in 0.1% Triton X-100 (blocking solution) for 30 min. Cells were incubated with primary antibodies diluted in blocking solution (anti-MAP2, 1:500, chicken, ab5392, Abcam; and anti-vGlut1 1:1000, guinea pig, AB5905, Merck Millipore) for 2 hr at room temperature (RT). After washing with PBS, the cells incubated with secondary antibodies in blocking solution for 1 hr at RT in the dark (anti-chicken Alexa 568, 1:1000, A11041, Thermo Fisher Scientific; and anti-guinea pig Alexa 647, 1:1000, A-21450, Thermo Fisher Scientific) and washed again. Coverslips were mounted with FluorSave and imaged on Zeiss CellObserver spinning disc confocal microscope (×40 water immersion objective; NA 1.2) with Zeiss Zen Blue 2012 software. Images were acquired as Z-stack and 9 images per plane to capture the whole island in the field of view. The images were post-processed with Zeiss Zen Black software and neuronal morphology was analyzed using SynD automated image analysis (Schmitz et al., 2011 (link)).

Kádková A., Murach J., Østergaard M., Malsam A., Malsam J., Lolicato F., Nickel W., Söllner T.H, & Sørensen J.B. (2024). SNAP25 disease mutations change the energy landscape for synaptic exocytosis due to aberrant SNARE interactions. eLife, 12, RP88619.

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Antibodies for Western Blot and Immunostaining

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Primary antibodies used were: mouse anti-GAPDH (G8795, Sigma-Aldrich; 1:10000 for western blot), mouse anti-c-Myc (M4439, Sigma-Aldrich; 1:6000 for western blot), mouse anti-Nav1.5 (S8809, Sigma-Aldrich; 1:2000 for western blot), rabbit anti-Nav1.5 (S0819, Sigma-Aldrich; 1:200 for immunostaining, 1:50 for STORM), chicken anti-Kir6.2 (C62; 1:50 for immunostaining and STORM), rabbit anti-Kir6.2 (Lee62; 1:50 for STORM), goat anti-Kir6.2 (N18, Santa Cruz; 1:500 for western blot), mouse anti-ankyrin-B (105/17, Neuromab; 1:2000 for western blot, 1:50 for STORM) and mouse anti-ankyrin-G (106/20, Neuromab; 1:2000 for western, 1:50 for STORM). Secondary antibodies used were donkey anti-mouse-HRP (715-035-150, Jackson, 1:10000), donkey anti-goat-HRP (705-035-147, Jackson, 1:10000), goat anti-chicken Alexa Fluor568 (A-11041, Thermo Scientific; 1:200), donkey anti-rabbit Alexa Fluor488 (711-545-152, Jackson; 1:200), and donkey anti-mouse Cy3 (715-165-151, Jackson; 1:200). All antibodies used in this study have been fully validated, either experimentally or in the literature. Details can be found in Figure 6—figure supplement 1 and Supplementary file 1.

Yang H.Q., Pérez-Hernández M., Sanchez-Alonso J., Shevchuk A., Gorelik J., Rothenberg E., Delmar M, & Coetzee W.A. (2020). Ankyrin-G mediates targeting of both Na+ and KATP channels to the rat cardiac intercalated disc. eLife, 9, e52373.

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Quantifying Cell Types in Nephron Segments

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Determination of the numbers of each cell type per unit length in microdissected PTs and CCDs from UNx and Sham mice was carried out using immunocytochemistry employing antibodies recognizing cell type specific markers, based on Purkerson et al.^{79 (link)}. The primary antibodies used were rabbit anti-AQP1 (LL266, in house, 1:100), mouse anti V-ATPase B1/B2 (F-6, sc-55544, Santa Cruz Biotechnology, Santa Cruz, CA, 1:100), anti-chicken AQP2 (CC 265, in house, 1:1000) and Alexa Fluor 568 phalloidin (A12380, Invitrogen, 1:400). The secondary antibodies were Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 488 goat anti-chicken, Alexa Fluor 568 goat anti-chicken and Alexa Fluor M-594 goat anti-mouse IgG. (A11034, A11039, A11041 and A11032, Invitrogen) each at 1:400 dilution. Cell nuclei were labelled with DAPI. Confocal fluorescence images were recorded with a Zeiss LSM780 confocal microscope using a 20× objective lens by Z-stack scanning. 3D images are reconstructed using z-stack files, and cell counting was performed on three-dimensional reconstructed tubule images using IMARIS Scientific Image Processing & Analysis software (v7.7.1, Bitplane, Zurich, Switzerland). Counting was automated using IMARIS “spot analysis” for nuclei. Tubule volume was calculated using IMARIS “surface analysis”.

Kikuchi H., Chou C.L., Yang C.R., Chen L., Jung H.J., Park E., Limbutara K., Carter B., Yang Z.H., Kun J.F., Remaley A.T, & Knepper M.A. (2023). Signaling mechanisms in renal compensatory hypertrophy revealed by multi-omics. Nature Communications, 14, 3481.

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Immunofluorescent Labeling of Brain Sections

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Brains were perfused in 4% PFA and post-fixed for 24 h and then kept in 0.01M PBS-0.05% sodium azide until further processing. Brain sections of 30 μm were cut and free-floating sections were blocked in an incubation solution of PBS, 0.25% BSA (BSA, A9418, Sigma-Aldrich), and 0.3% Triton X-100 (T8787, Sigma-Aldrich) with 4% normal goat serum (D9663, Sigma-Aldrich, RRID:AB_2810235) for 2h at room temperature (RT, 20–25°C). Then, sections were incubated with rabbit GFP antibody (1:500, A11122, Thermo Fisher Scientific Cat# A-11122, RRID:AB_221569) and chicken anti-vimentin (1:500; Millipore, catalog no. AB1620, RRID:AB_90774) in the same blocking buffer for 48h in agitation at 4°C. After PBS rinses, immunoreactivity was revealed with Alexa 488-conjugated secondary antibody goat anti-rabbit IgG (1:400, A11008, Invitrogen, RRID:AB_143165) and Alexa Fluor 568 conjugated secondary antibody goat anti-chicken IgG (1:4700, A11041; RRID: AB_144696) for 90 min. After Hoechst 33,258 (pentahydrate bis-benzimide, 1μg/mL, Invitrogen, RRID:AB_2651133) incubation for nuclei visualization, sections were mounted in slides and covered with mount containing Mowiol medium (Calbiochem, Merck Millipore).

Bakker W., Imbernon M., Salinas C.G., Moro Chao D.H., Hassouna R., Morel C., Martin C., Leger C., Denis R.G., Castel J., Peter A., Heni M., Maetzler W., Nielsen H.S., Duquenne M., Schwaninger M., Lundh S., Johan Hogendorf W.F., Gangarossa G., Secher A., Hecksher-Sørensen J., Pedersen T.Å., Prevot V, & Luquet S. (2022). Acute changes in systemic glycemia gate access and action of GLP-1R agonist on brain structures controlling energy homeostasis. Cell Reports, 41(8), 111698.

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Quantifying pS65-Ub Levels via Imaging

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To quantify pS65‐Ub levels using automated high‐content imaging, fibroblasts were seeded in 96‐well imaging plates (Fisher Scientific, 08772225) and allowed to attach overnight. Cells were then treated for 0, 4, 8, or 24 h with 1 μM valinomycin and fixed in 4% paraformaldehyde after one wash with PBS. Fibroblasts were immunostained with primary antibodies against pS65‐Ub (Cell Signaling Technology, 62802; 1:1250) and HSP60 (arigo Biolaboratories, ARG10757; 1:2000) followed by incubation with secondary antibody (Invitrogen, A‐11034 and A‐11041; 1:1000) and Hoechst 33342 (Invitrogen, H21492; 1:5000). Plates were imaged on a BD Pathway 855 (BD Biosciences, San Jose, CA, USA) with a 20× objective using a 2 × 2 montage (no gaps) with laser autofocus every second frame as previously described [33 (link)]. Raw images were processed using the built‐in AttoVision V1.6 software. Regions of interest were defined as nucleus and cytoplasm using the built‐in “RING‐2 outputs” segmentation for the Hoechst channel after applying a shading algorithm. Values were normalized to 0 h and 24 h valinomycin treated control cells as 0% and 100%, respectively.

Hou X., Chen T.H., Koga S., Bredenberg J.M., Faroqi A.H., Delenclos M., Bu G., Wszolek Z.K., Carr J.A., Ross O.A., McLean P.J., Murray M.E., Dickson D.W., Fiesel F.C, & Springer W. (2023). Alpha‐synuclein‐associated changes in PINK1‐PRKN‐mediated mitophagy are disease context dependent. Brain Pathology, 33(5), e13175.

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Immunofluorescent Staining of Larval Tissues

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Developmentally staged larvae (after euthanisation in MS222) were fixed in 4% paraformaldehyde (1 h), dehydrated to 100% methanol, and stored at −20 °C before staining. Immunolabeling was done as previously described [21 (link)]. Primary antibodies were anti-Smad9 (ab96698, Abcam, Cambridge, MA, USA) used at a 1:200 dilution, anti-Col2a1 (II-II6B3, DSHB, University of Iowa, Iowa, IA, USA) used at 1:20 and anti-GFP (chicken polyclonal, Abcam, ab13970) used at a 1:300 dilution in blocking buffer (5% horse serum). Additionally, rabbit anti-Smad1 (1:100 dilution, sc-6031-R, Santa Cruz, Dallas, TX, USA), rabbit anti-Smad5 (1:200 dilution, ab227090, Abcam), and rat anti-mCherry [16D7] (1:100 dilution, M11217, Invitrogen, Carlsbad, CA, USA). Primary antibodies were used in 5% horse serum in 0.2% PBS-Triton-X100 blocking buffer. Secondary antibodies Alexa Fluor 488 anti-rabbit, Alexa Fluor 568 anti-chicken, Alexa Fluor 647 anti-mouse (A21206, A11041, and A31571 respectively, Invitrogen, Carlsbad, CA, USA), and Dylight 650 anti-Rat (SA5-10029, ThermoFisher, Waltham, MA, USA) were used at a 1:500 dilution. Samples were mounted in 1% low melting point agarose and imaged as described below.

McDonald G.L., Wang M., Hammond C.L, & Bergen D.J. (2021). Pharmacological Manipulation of Early Zebrafish Skeletal Development Shows an Important Role for Smad9 in Control of Skeletal Progenitor Populations. Biomolecules, 11(2), 277.

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