Transverse sections were pre-treated with 50% methanol for 20 min at 22–24°C, washed in tris-buffered saline (TBS) and blocked for 1 hr in TBS containing 5% normal donkey serum and 0.25% Triton X-100. Sections were incubated in streptavidin 488 (1:400, Life Tech) and/or primary antibodies against serotonin (1:5000, Immunostar), synaptophysin (1:300, Sigma), IBA1 (1:1500, Wako), GFAP (1:1500, Encor Bio). Sections were washed with TBS and then incubated in Alexa-Fluor 488- or 594-conjugated secondary antibodies (Thermo Fisher, 1:500) for 1 hour. Sections were washed with TBS, mounted on slides, and coverslipped with Mowiol mounting medium (http://cshprotocols.cshlp.org/content/2006/1/pdb.rec10255 ). All antibodies were used previously in monkeys [30 (link)].
Alexa fluor 488 or 594 conjugated secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
Alexa Fluor 488- or 594-conjugated secondary antibodies are fluorescent dye-labeled antibodies that can be used for detection and visualization in immunoassays and other applications. These antibodies target and bind to primary antibodies, allowing for the indirect detection of target proteins or other molecules of interest.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Bz 9000, by Keyence (5 mentions)
Lsm 510 meta, by Zeiss (3 mentions)
Synaptophysin, by Merck Group (2 mentions)
Streptavidin 488, by Thermo Fisher Scientific (2 mentions)
Serotonin, by Immunostar (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Antibody diluent, by Agilent Technologies (2 mentions)
Fv1000, by Olympus (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Ab13970, by Abcam (2 mentions)
Prolong gold mounting reagent, by Thermo Fisher Scientific (2 mentions)
Ab5733, by Merck Group (2 mentions)
Mouse anti smooth muscle actin, by Merck Group (2 mentions)
Anti f4 80, by BD (2 mentions)
Tcs sp8 confocal microscope, by Leica (2 mentions)
Prolong gold antifade mounting medium with dapi, by Thermo Fisher Scientific (1 mentions)
87 protocols using alexa fluor 488 or 594 conjugated secondary antibody
1
Immunohistochemical Staining of Brain Tissue
2
Immunohistochemical Analysis of Retinal Cells
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Eyeballs were collected and fixed in 4% paraformaldehyde (PFA) at 4°C overnight. Next day, retinas were dissected, washed with PBS, blocked and permeabilized with PBS containing 5% normal goat serum and 0.3% Triton X-100 for 3 hours. Subsequently, retinas were incubated with primary antibody against Tuj1 (1:400, BioLegend, San Diego, CA, USA) or CD45 (1:400, BD Biosciences, San Jose, CA, USA) at 4°C overnight. After washing, retinas were then incubated with appropriate AlexaFluor 488 or 594-conjugated secondary antibodies (1:400, Thermo Fisher Scientific, Waltham, MA, USA) at 4°C for 4 hours. Finally, retinas were mounted and images were captured with confocal microscopy (LSM 510 Meta, Carl Zeiss, Inc., Thornwood, NY, USA).
3
Immunofluorescence Staining of Corneal Samples
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Transverse sections (5 µm thick) of ex vivo or organ-cultured corneas were fixed in 1% vol./vol. formalin or methanol for 5–10 min. LEC cultures were fixed in 10% formalin, permeabilised in 0.2–0.5% Triton X-100 (MilliporeSigma) and blocked in 5% vol./vol. normal goat serum [20 (link)]. Primary antibodies are described in ESM Table 3 . AlexaFluor 488- or 594-conjugated secondary antibodies were from Thermo Fisher. All antibodies were diluted in PBS. Slides were mounted using ProLong Gold Antifade mounting medium with DAPI (Thermo Fisher). For each marker, the same exposure time was used when photographing stained sections or cells. Negative controls without a primary antibody were routinely included. Changes in marker expression in individual cases of organ-cultured corneas are indicated in ESM Table 4 .
4
Immunohistochemical Staining of Brain Tissue
5
3D Culture Immunofluorescence Assay
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Alexa Fluor 488 or 594 conjugated secondary antibodies (Thermo Fisher Scientific, MA, US) were used for 3D culture immunofluorescence assays. MCF10A-PI3KαH1047R 3D-acini were fixed (4% PFA) at day 15 and processed for immunofluorescence microscopy analysis as established previously [59 (link)]. Confocal analyses were performed using the Leica SP5 Multi-photon confocal microscope equipped with UV diode (405 nm), argon (458, 476,488 and 514 nm), HeNe (543 nm) and a HeNe (633 nm) lasers. All images were obtained with a x63 objective. Quantitative measurements of optical density were performed with ImageJ (National Institutes of Health, US). Acinar size was calculated with LAS X software (Leica) following the equation [(length × width2)/2 = acini volume (mm3)] and plotted with GraphPad Prism.
6
Immunofluorescence Staining of NIH/3T3 Cells
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NIH/3T3 cells were plated at 2.5 × 105 cells per well in 6-well plates with coverslips and cultured for 24 h. The cells were fixed with 3.7% formaldehyde for 10 min at room temperature. After washing with PBS, the cells were treated with ice-cold methanol for 10 min at −30 °C. After blocking with Blocking One Histo (Nacalai Tesque, Kyoto, Japan) and washing with PBS containing 0.05% Tween® 20, the cells were incubated with primary antibodies overnight at 4 °C. After washing with PBS, Alexa Fluor® 488- or 594-conjugated secondary antibodies (Thermo Fisher Scientific) were used. Nuclei were stained using 0.1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Dojindo, Kumamoto, Japan). The fluorescence was observed by a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and a Leica TCS SP8 microscope (Leica Microsystems, Wetzlar, Germany).
7
Immunocytochemical Analysis of EGFR and CD44 in MDA-MB-231 Cells
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Immunocytochemistry was conducted to assess the expression and co-localization of EGFR and CD44 in MDA-MB-231 cells. Cells in Falcon® chambered cell culture slides (BD Biosciences) were fixed with 4% paraformaldehyde, washed with PBS, and incubated with 0.2% Triton X-100 for 13 min. Primary antibodies were applied to the cells in antibody-diluent (Dako, Denmark) were incubated overnight at 4°C. For secondary antibody reactions, Alexa Fluor®-488 or -594 conjugated secondary antibodies (Thermo Fisher Scientific) were used for staining and then mounted with ProLong® Gold Antifade Reagent with DAPI (Thermo Fisher Scientific). Imaging of the cells was performed using a Carl Zeiss confocal microscope (Weimar, Germany), and the intensity of the EGFR and CD44 signal was analyzed using the intensity profile tool.
8
Immunofluorescence Staining of Cultured Cells
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Cells were seeded into 8-well chambered slides (Thermo Fisher Scientific). After treatment, cells were fixed with 4% paraformaldehyde at 37 °C for 30 min, permeabilized with 0.2% Triton X-100 and incubated in 3% BSA blocking solution for 30 min to block non-specific bindings of antibody. Cells were then incubated with a primary antibody diluted in 3% BSA in PBS overnight at 4 °C. Then, the cells were washed with PBS for 5 min 3 times and incubated with Alexa Fluor 488- or 594-conjugated-secondary antibodies (Thermo fisher Scientific) for 1 h at room temperature in the dark. Cell nuclei were stained with 1 μg/ml Hoechst33342 for 30 min. The fluorescence images were acquired by Zeiss LSM 880 Confocal system (Carl Zeiss, Thornwood, NY).
9
Spinal Cord Immunohistochemistry in Mice
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Mice were anesthetized and perfused intracardially with saline and 4% paraformaldehyde in 0.1 M phosphate buffer. L4-5 spinal tissues were collected, fixed in 4% paraformaldehyde overnight at 4°C, then transferred to 30% sucrose in 0.1 M phosphate buffer at 4°C. After that, specimens were embedded by embedded medium (Cat# 4583, Sakura). Subsequently, 30-μm-thick serial sections were prepared using a freezing microtome and blocked with 1% bovine serum albumin (Cat# MB4219, Meilunbio) for 45 min. Spinal sections were then incubated overnight at 4°C with the following primary antibodies: antibody against translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20) (1: 250, Cat# ab186735, Abcam), antibody against neuronal nuclei (NeuN) (1: 500, Cat# ab104224, Abcam), anti-Iba-1 (1: 500, Cat# ab48004, Abcam). After washing, free-floating sections were incubated for 2 h at room temperature with the corresponding Alexa Fluor 488- or 594-conjugated secondary antibodies (1: 500, Cat# A-21,207 and A-21,202, Thermo Fisher Scientific). Fluorescence images were captured using a confocal scanning laser microscope (FV1000, Olympus), and images were shown as merged Z-stack projections consisting of approximately 10 optical slices (1 μm per slice). The number of Iba-1 positive cells and the count of TOMM20 positive neurons and microglia were measured using ImageJ software.
10
Imaging ARPE-19 Cells' Cytoskeleton
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ARPE-19 cells were seeded in four-well glass cell-culture slides (SPL Life Sciences, Pocheon, Korea). The RPE cells were cultured in the presence or absence of 20 nM bortezomib at 24 h after plating and cultured to 50–60% confluence. After the medium was removed followed by three rinses with PBS, the cells were fixed with 4% paraformaldehyde for 30 min. For observations of filamentous actin, the cells were stained with Alexa Fluor 488–conjugated phalloidin/Vectashield mounting solution containing DAPI. For the immunocytochemical analyses, the cells were permeabilized with 0.4% (v/v) Triton X-100 for 15 min, and then blocked with 1% (w/v) bovine serum albumin (BSA) in 0.1% (v/v) Tween-20/PBS for 30 min. Samples were incubated with primary antibodies for 12 h at 4 °C, followed by Alexa Fluor 488– or 594–conjugated secondary antibodies (Thermo Fisher Scientific) for 2 h at room temperature. The nuclei were stained with DAPI after several rinses. Each slide was observed on an inverted fluorescent microscope (Ti-U, Nikon Instruments Inc., Melville, NY), and images were taken using the NIS-Elements software (Nikon Instruments Inc.).
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