Third-instar larvae were dissected, fixed in Bouin’s fixative or 4% PFA in PBS, and immunostained as previously described (Eaton et al., 2002 (link); Harris et al., 2015 (link)). Dissected third instar larvae were fixed with PFA (4%) and incubated overnight at 4 C with primary antibodies (mouse anti Flag 1:50; rabbit anti-RFP 1:100; rabbit anti-Dlg, 1:1000; anti-Syt1 1:1000, anti-Brp 1:100, Life Technologies). Alexa-conjugated secondary antibodies and goat anti-HRP were used (Jackson Laboratories 1:500). Images were acquired with either a Zeiss LSM700 confocal microscope equipped with Zen software using a 63X 1.6 NA oil immersion objective or an upright epifluorescence deconvolution confocal microscope (Axiovert 200, Zeiss) equipped with a 100X objective (N.A. 1.4), cooled CCD camera (CoolSnap HQ, Roper Scientific). Slidebook 5.0 (3I, Intelligent Imaging) was used for capturing, deconvolving and analyzing images. Structured illumination microscopy (Nikon LSM 710 equipped with 63X objective and Andor Ixon EMCCD camera) was used to perform Brp-GFP and MCTP-Flag colocalization experiments. Bouton numbers and Brp numbers and densities were quantified as described previously (Harris et al., 2015 (link)).
Alexa conjugated secondary antibodies
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa-conjugated secondary antibodies are fluorescently labeled antibodies that bind to the primary antibody. They are designed to be used in a variety of immunoassay techniques, such as immunofluorescence and flow cytometry, to detect and visualize specific target proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Cryostat, by Leica camera (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Tissue tek oct compound, by Sakura Finetek (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Coolsnap hq, by Teledyne (2 mentions)
Anti brp, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Oxford Instruments (2 mentions)
Emccd camera, by Oxford Instruments (2 mentions)
Axiovert 200, by Zeiss (2 mentions)
Apotome ax10, by Zeiss (2 mentions)
Rabbit anti phospho histone h3 ser10 antibody, by Merck Group (2 mentions)
Recombinant human wnt3a, by R&D Systems (2 mentions)
Rabbit anti ankyrin g antibody, by Santa Cruz Biotechnology (2 mentions)
H 102, by Santa Cruz Biotechnology (2 mentions)
Mouse anti zo 1 antibody, by Thermo Fisher Scientific (2 mentions)
Chicken anti gfp antibody gfp 1020, by Aves Labs (2 mentions)
Mouse anti gsk3β antibody, by BD (2 mentions)
Mouse anti py216 gsk3β antibody, by Abcam (2 mentions)
Mouse anti e cadherin antibody, by BD (2 mentions)
5 bromo 2 deoxyuridine brdu, by Merck Group (2 mentions)
28 protocols using alexa conjugated secondary antibodies
1
Immunostaining of Drosophila Larval Neurons
2
Drosophila Neuromuscular Junction Immunostaining
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Third-instar larvae were dissected, fixed in Bouin’s fixative or 4% PFA in PBS, and immunostained with previously described methods (Eaton et al., 2002 (link); Harris et al., 2015 (link)). Third instar larvae were dissected with cold HL3 and immediately fixed with PFA (4%) and incubated overnight at 4 C with primary antibodies (rabbit anti-Dlg, 1:1000; anti-Brp 1:100, Life Technologies). Alexa-conjugated secondary antibodies were used for secondary staining (Jackson Laboratories 1:500). An inverted epifluorescence deconvolution confocal microscope (Axiovert 200, Zeiss) equipped with a 100X objective (N.A. 1.4), cooled CCD camera (CoolSnap HQ, Roper Scientific) was used to acquire images. All acquisition, deconvolution and analysis were done by Slidebook 5.0 software (3I, Intelligent Imaging). Structured illumination microscopy (Nikon LSM 710 equipped with 63X objective and Andor Ixon EMCCD camera) was used to perform Brp puncta and Dlg labeling experiments. Bouton numbers were quantified as described previously (Harris et al., 2015 (link)).
3
Retinal Immunofluorescence Staining Protocol
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Animals were anaesthetized under isoflurane and sacrificed by cervical dislocation or decapitation for early post-natal pups. Preparation and immunofluorescence staining of retinae and hyaloid vessels were as described previously6 (link). For phospho-T53-DAT (pDAT) quantification, retinae of each genotype and light condition were collected in dim red light (dark-adapted) or normal light from at least 3 different induction experiments and mounted in the same OCT blocks. Retinal sections were processed, stained, and imaged together to compensate for batch differences. Alexa-conjugated secondary antibodies were purchased from Jackson ImmunoResearch (JIR). Images were captured using Zeiss ApoTome AX10 or Zeiss LSM700 confocal microscopes and processed by ImageJ (NIH) and Adobe Photoshop (Adobe Systems). Primary antibodies and lectins used in this study are listed in Supplementary Table 1 .
4
Cardiac Tissue Cryosectioning and Immunostaining
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Hearts were flash frozen in liquid nitrogen cooled isopentane and cryosectioned at 10 μm. Phalloidin (Alexa Fluor 594, Life Technologies, cat. A12381) and DAPI (Life Technologies, cat. D1306) stained sections were used to identify the scars. Sections were fixed in 100% acetone at −20 °C for 5 min then blocked for one hour in blocking solution containing (in mM) 20 Tris pH 7.4, 155 NaCl, 2 EGTA, 2 MgCl2 and 5% v/v Donkey Serum (Jackson Immunoresearch). Primary antibodies were incubated overnight at 4 °C or one hour at room temperature. Primary antibodies were diluted in blocking buffer as follows: Mouse anti-beta galactosidase (Promega, cat. Z3781) 1:100, chicken anti-Vimentin (Abcam, cat. 24525), rabbit anti-Cx43 (Millipore, cat. AB1727). Alexa conjugated secondary antibodies (Jackson Immunoresearch Laboratories) were used in a 1:200 dilution.
5
Immunohistochemistry Protocol for Cell Signaling
6
Multiparametric Immunostaining and Western Blotting
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The primary antibodies used in immunostaining were as follows: RNF20 (21615-1-AP; Proteintech, Rosemont, IL, USA), NESTIN (MAB353; Millipore, Darmstadt, Germany), ACSBG1 (ab118154; Abcam, Cambridge, UK), GFAP (G6171; Sigma, St. Louis, MO, USA), GFAP (Z0334 Dako, Santa Clara, CA, USA), GFAP (60190-1-Ig; Proteintech), GLAST (BMP009; MBL, Beijing, China), Tenascin C (110-68136; NB, Littleton, CO, USA), Ki67 (ab137876; Abcam), FLAG (F1804; Sigma), STAT3 (#9139; Cell Signaling Technology, Beverly, MA, USA). The primary antibodies used in western blotting were as follows: RNF20 (21615-1-AP; Proteintech), GLAST (20785-1-AP; Proteintech), ACSBG1 (ab118154; Abcam), GFAP (G6171; Sigma), STAT3 (#9139; Cell Signaling Technology), phospho-STAT3 (#9145; Cell Signaling Technology), Ubiquityl-Histone H2B (Lys 120) (#5546; Cell Signaling Technology), H4K16ac (07-329; Millipore), H3K9me3 (07-442; Millipore), Trimethyl-Histone H3 (Lys 27) (07-449; Millipore), H2B (ab1790; Abcam), FLAG (F1804; Sigma), HA (M20003; Abmart, Shanghai, China), β-ACTIN (20536-1-AP; Proteintech). Alexa-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were used for immunostaining. IR Dye (LI-COR Biosciences, Lincoln, Nebraska, USA) 800 CW donkey anti-rabbit and 680 CW donkey anti-mouse secondary antibodies were used in western blotting.
7
Immunostaining of Nfasc155 and Nfasc186 in HEK Cells
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Human embryonic kidney (HEK) cells were transfected with Nfasc155 or Nfasc186 variants using JetPEI (Polyplus-transfection). After 48 h, cells were incubated for 20 min at 37°C with rabbit antibodies against Nfasc186 (D6G60; Cell Signaling Technology) or Nfasc155 (D7B6O; Cell Signaling Technology) diluted 1/50 in Opti-MEM™ or with 1 µg of gliomedin-Fc (Devaux, 2012 (link)) diluted in Opti-MEM™ (ThermoFisher Scientific). Cells were then washed in phosphate-buffered saline (PBS), fixed with 2% paraformaldehyde in PBS and blocked with blocking solution (5% fish skin gelatin containing, 0.1% Triton™ X-100 in PBS). HEK cells were incubated with a mouse anti-Myc antibody (Roche; 1/200) for 1 h, then incubated with Alexa conjugated secondary antibodies (1/500; Jackson ImmunoResearch) for 30 min. Coverslips were washed three times in PBS, stained with DAPI, and mounted with Mowiol® plus 2% DABCO. In some experiments, HEK cells were co-transfected with GFP and Nfasc155 variants. In those experiments, cells were fixed and revealed with a mouse anti-Myc antibody. Immunostainings were examined using an ApoTome fluorescence microscope (ApoTome, AxioObserver and AxioCam MRm, Carl Zeiss MicroImaging GmbH). Digital images were manipulated into figures with CorelDRAW and Corel Photo-Paint.
8
Immunostaining and Confocal Imaging of Zebrafish and Mouse Pancreata
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Zebrafish embryos and mouse pancreata were fixed and immunostained as described [30 (link)].
Primary antibodies (Supplementary Table 2 ) were detected with 1:500 dilutions of Alexa-conjugated secondary antibodies (Jackson ImmunoResearch). Confocal imaging was performed using a Zeiss LSM700 microscope and quantified by measuring pixel density per insulin-positive cell (Fiji software). To measure nuclear vs. cytoplasmic intensity of markers in cells, a DAPI signal was used as a mask to quantify only pixel density within the nucleus. To quantify Insulin+ glucagon+ polyhormonal cells, all visible individual islet cells that exhibited both insulin staining and glucagon staining were counted.
Primary antibodies (
9
Immunohistochemistry Protocol for Cell Signaling
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The following primary antibodies were used in this study: rabbit anti-ankyrin G antibody (H-215, Santa Cruz Biotechnology), rabbit anti-β-catenin antibody (H-102, Santa Cruz Biotechnology), mouse anti-β-catenin antibody (610153, BD Transduction Laboratories), mouse anti-ZO-1 antibody (33–9100, Invitrogen), chicken anti-GFP antibody (GFP-1020, Aves Labs), mouse anti-BrdU antibody (M0744, Clone Bu20a, DakoCytomation), rabbit anti-Ki67 antibody (Clone SP6, Lab Vision/Thermo Scientific), rabbit anti-phospho-Histone H3 (Ser10) antibody (06–570, Millipore), mouse anti-GSK3β antibody (610202, BD Transduction), mouse anti-pY216 GSK3β antibody (ab75745, Abcam), anti-actin (Sigma A5316, clone AC-74) and mouse anti-E-cadherin antibody (610181, BD Transduction). Alexa-conjugated secondary antibodies (Jackson ImmunoResearch) were used for IHC and ICC. Recombinant human Wnt-3a was purchased from R&D Systems (Catalog number: 5036-WN). BrdU (5-Bromo-2’-deoxyuridine) was purchased from Sigma-Aldrich (B5002-5G).
10
Immunostaining of Retinal Sections
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Cell cultures on the coverslips were fixed for 20 min with 4% PF and the rat retinal sections were recovered to RT. The coverslips or retinal sections were washed three times for 5 min in ice-cold 0.01 M PBS. Subsequently, the coverslips or retinal sections were blocked for 1 h in blocking buffer, i.e., PBS containing 5% normal bovine serum and 0.3% Triton X-100, and then incubated with combinations of the primary antibodies against the following targets, overnight at 4°C: tAIF (1:500, sc-13116, Santa Cruz), CAST (1:50, sc-2-7799, Santa Cruz, Dallas, TX, United States) and calpain2 (1:100, ab39165, Abcam, Cambridge, United Kingdom). The coverslips or retinal sections were shifted to RT for 30 min, washed three times as described above, and incubated with Alexa-conjugated secondary antibodies (1:200, Jackson Immuno Research, West Grove, PA, United States) for 2 h. After washing three times in PBS, the coverslips or retinal sections were covered with Vectashield mounting medium containing DAPI. The coverslips or retinal sections were stained in parallel and images were acquired, with a fluorescence microscope, using the same settings.
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