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.
A31556
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A31556 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for performing a specific function within a laboratory setting. The core function of this product is to provide a reliable and accurate solution for a particular laboratory application. However, without access to detailed product information, I cannot provide a more specific description while maintaining an unbiased and factual approach. The intended use and detailed specifications of this product are not available to me.
Automatically generated - may contain errors
Lab products found in correlation
A11029, by Thermo Fisher Scientific (2 mentions)
A21236, by Thermo Fisher Scientific (2 mentions)
F7425, by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Ab290, by Abcam (1 mentions)
A11031, by Thermo Fisher Scientific (1 mentions)
A11041, by Thermo Fisher Scientific (1 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
Alexa 405, by Thermo Fisher Scientific (1 mentions)
A11003, by Thermo Fisher Scientific (1 mentions)
Anti glua1, by Merck Group (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Alexa 350, by Thermo Fisher Scientific (1 mentions)
Alexa 546, by Thermo Fisher Scientific (1 mentions)
Ab5622, by Merck Group (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Mouse α flag, by Merck Group (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
A 21235, by Thermo Fisher Scientific (1 mentions)
Fluoroshield mounting medium, by Abcam (1 mentions)
10 protocols using a31556
1
Immunofluorescence and Western Blot Protocols
2
Quantifying NMDA Receptor Signaling
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NMDA was purchased from Tocris Bioscience. Commercial antibodies were as follows: anti-GluA1 (04-855, Millipore), anti-GluA1 (SAB5201086, Sigma), anti phospho-GluA1 (Ser831) (36-8200, Invitrogen), anti-phospho-GluA1 (Ser845) (36-8300, Invitrogen), anti-stargazin (C8206, Sigma), anti-4.1N (276103, Synaptic Systems), anti-GST (RPN1236, Amersham), anti-HA, (901501, Covance), anti-FLAG (F7425, Sigma), and anti-MAP-2 (AB5622, Millipore) antibodies; Alexa 350 (A-11045, Thermo Fisher), Alexa 405 (A-31556, Thermo Fisher), Alexa 488 (A-11008, Thermo Fisher), Alexa 546 (A-11003, Thermo Fisher), HRP (18-8816-33, 18-8817-33, Rockland) conjugated secondary antibodies, and pre-immune IgG (CYP450-GP HU-A000).
3
Immunofluorescence Analysis of Autophagy Markers
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Immunofluorescence analysis was performed as described previously (Carroll et al, 2018 (link)). In brief, cells seeded on coverslips in 24‐well plates were fixed in 3.7% formaldehyde in PBS for 7 min at room temperature and permeabilised in methanol for 4 min at −20°C. Cells were then blocked for 1 h in 5% normal goat serum (Sigma‐Aldrich) in PBS at room temperature and incubated with primary antibodies overnight at 4°C. Cells were washed three times and incubated with the appropriate secondary antibodies for 1 h at room temperature (Thermo Fisher Scientific, A31556 and A21235, 1:1,000). Cells were washed, and coverslips were mounted on slides with fluoroshield mounting medium (Abcam). Fluorescence images were obtained described as above. The number of puncta colocalised with NDP52 or with YFP‐Parkin per cell was quantified. The following primary antibodies were used: mouse α‐Flag (Sigma‐Aldrich, F3165, 1:1,000, for NDP52 staining), rabbit α‐LC3 (CST, 3868S, 1:250), rabbit α‐Atg13 (CST, 13468S, 1:100) and rabbit α‐Atg16L (CST, 8089S, 1:100).
4
Click Labeling and Immunostaining of Cells
5
Pericyte Visualization and Kidney Injury Assessment
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Pericytes were labelled by expression of DsRed under control of the NG2 promoter (in mice), or with antibodies to NG2 (1:200; Abcam ab50009, Cambridge, United Kingdom), α-smooth muscle actin (α-SMA) (1:100; Abcam ab5694, Cambridge, United Kingdom), or myosin light chain (phospho S20, 1:100, Abcam ab2480, Cambridge, United Kingdom), and the capillary basement membrane and pericytes were labelledwith isolectin B4-Alexa Fluor 647 (1:200, overnight; Molecular Probes, I32450, Thermo Fisher Scientific, Waltham, MA). Z-stacks of the cortex and outer medulla (frame size 640.17 × 640.17 µm) for cell counting were acquired confocally (Zeiss LSM 700, Oberkochen, Germany). Pericyte intersoma distance was calculated between pairs of pericytes on capillaries within the same imaging plane. Kidney damage was assessed using kidney injury molecule-1 (Kim-1) antibody (1:100, overnight; Novus Biologicals, NBP1-76701, Abingdon, United Kingdom). Red blood cells were labelled with antibody to glycophorin A (1:2000, AbCam ab9520, Cambridge, United Kingdom). Alexa Fluor conjugated secondary antibodies were added overnight (1:500; ThermoFisher, A31572, A31556, A31570, Waltham, MA).
6
Quantitative Cardiac Tissue Analysis
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Paraffin-embedded LV sections were treated as described in the previous section for deparaffinization, antigen retrieval, and nonspecific binding blockade. Subsequently, samples were incubated overnight at 4 °C with a vimentin primary antibody (Ab92547 Abcam, Cambridge, UK; 1/150 dilution) and the next morning with the secondary antibody goat antirabbit IgG conjugated to Alexa Fluor 405 (A31556 Thermo Fisher, Waltham, MA, USA; 1/200 dilution) at room temperature for 120 min. Additionally, samples were labeled with a wheat germ agglutinin (WGA) lectin conjugated to Alexa Fluor 594 (W11262 Thermo Fisher; 1/200 dilution) and with isolectin GS-IB4 conjugated to Alexa Fluor 488 (I21411 Thermo Fisher; 1/200 dilution) for 120 min. Sections were mounted with Fluoromount (Sigma-Aldrich, St. Louis, MO, USA). Microphotographs were obtained in three different regions at the three different bandpass filters (345–445, 470–525, and 550–605) for both LV and IVS at a magnification of 63×. Myocyte count, cross-sectional area, extracellular matrix content (ECM), capillary, and fibroblast count were assessed with morphological measurements with ImageJ [13 (link)].
7
Combining RNA FISH and EVA Imaging
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To combine RNA FISH with EVA, coverslips after biotin-tyramide TSA reaction were incubated in hybridization washing solution for 1 h at RT, excess of solution was drained on paper towel, coverslips were placed on 10 μl of hybridization solution containing EVA oligo mix on a slide, sealed with rubber cement, and processed according to the EVA protocol as described above. Biotin was visualized using rabbit anti-biotin antibodies (1:300, A150-109A, Bethyl) and goat anti-rabbit Alexa Fluor 405 IgGs (1:300, A31556, Thermo Fisher).
8
Optogenetic Calcium Imaging in HeLa Cells
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Hela cells were transfected with pCAG-CaMAPRI2 and a vector expressing the ATP-gated Ca2+ channel P2X using the manufacturer’s protocols (Lipofectamine 2000, Invitrogen). After 24 h, cells were washed with Hank’s Balanced Salt Solution, and ATP was administered to a final concentration of 100 μM immediately followed by PC of one field of view (20 s of 400-nm light at 3 mW cm−2) on a Nikon Ti Eclipse wide-field microscope equipped with an LED illuminator (SPECTRA-X, Lumencor), a ×20 objective and an sCMOS camera (Zyla, Andor). The cells were then fixed using formaldehyde (4% in phosphate-buffered saline (PBS) containing 10 mM EGTA, 10 min) and immunostained using standard protocols with anti-CaMPARI-red (1:10,000) and rabbit anti-FLAG (1:3000, F7425, Sigma-Aldrich) as primary and goat-anti-rabbit Alexa Fluor 405 (1:1000; A31556, Invitrogen) and goat-anti-mouse Alexa Fluor 647 (1:1000, A21236, Invitrogen) as secondary antibodies. Cells were then imaged on the same microscope using the 4,6-diamidino-2-phenylindole, fluorescein isothiocyanate, tetramethylrhodamine, and Cy5 imaging channel. In the resulting four-color image, individual cells were segmented in the green channel and four color intensities were calculated.
9
Quantifying Parasite Ubiquitination in Macrophages
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150,000 BMDMs per well were seeded in an 8-well μ-slide and stimulated with 10 ng/mL (~100 U/mL) recombinant mouse IFNγ for 24 h prior to infection. The BMDMs were infected with parasite strains at an MOI of 0.3 for 3 h, then washed and fixed with 4% w/v formaldehyde for 15 min. Prior to permeabilisation, the cells were blocked with 2% w/v BSA for 1 h and extracellular parasites were stained with 1:1000 rabbit anti-T. gondii (Abcam #ab138698) for 1 h at room temperature followed by 1:1000 goat anti-rabbit 405 (Invitrogen #A31556) for 1 h at room temperature. The cells were then permeabilsed with 0.2% v/v Triton X-100 for 15 minutes, blocked again with 2% w/v BSA for 1 h, stained with 1:200 mouse anti-ubiquitinylated proteins (Sigma #04–263) overnight at 4°C, and finally stained with 1:1000 goat anti-mouse 488 (Invitrogen #A11029) for 1 h at room temperature. Nine tiled fields of view were captured for each well on a Nikon Ti-E inverted widefield fluorescence microscope as above. The images were blinded, and the percentage of ubiquitinated vacuoles was determined manually using ImageJ, excluding T. gondii cells which were positive for extracellular staining. A median of 290 vacuoles were analysed per strain per replicate. Differences between strains were determined by two-sided t-test with Benjamini-Hochberg adjustment.
10
Antibody Immunofluorescence Staining Protocol
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Rabbit polyclonal antibodies against L. monocytogenes were a gift from Dr. Pascale Cossart (Institut Pasteur), mouse monoclonal antibodies against GFP were from Invitrogen (A-11120); antibodies against mouse p62-Ick ligand were from BD Biosciences (610832), rat polyclonal antibodies against LAMP1 were from Developmental Studies Hybridoma (1D4B) and antibodies against mono- and poly-ubiquitinated protein were from Biomol International (FK2; BML-PW8810-0500). All fluorescent secondary antibodies—goat anti rabbit 405, goat anti rat Cy3, goat anti mouse 568, goat anti mouse 488—were AlexaFluor conjugates from Molecular Probes (A31556; A10522, A11004; A11029; Invitrogen).
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