The immunofluorescence assay was performed as described [33 (link)]. Flies were fed with 5% sucrose solution with or without rotenone for 12 h. Intestines were dissected in PBS and fixed with 4% paraformaldehyde for 25 min at room temperature. The samples were blocked with a blocking solution (PBS with 0.3% Triton X-100, 0.2% goat serum and 0.1% fetal calf serum) for 25 min, incubated in primary antibodies overnight at 4 °C, and washed in PBS supplemented with 0.3% Triton X-100 (PBST). Then the samples were incubated with the secondary antibodies conjugated with Alexa 488 or 568 (Thermo Fisher, 1:1000, Waltham, MA) for 2 h at room temperature. Primary antibodies were α-syn (Novus Biologicals, 1:1,000, Centennial, CO), phospho-histone 3 (Millipore, 1:1,000, Burlington, MA), Dlg (DHSB, 1:50, Iowa City, IA) and phospho-Jun N-terminal Kinase (phospho-JNK) (Millipore, 1:200), while nuclear DNA was detected by DAPI (Thermo Fisher). All immunostained samples were mounted with Vectashield (Vector Laboratories, Newark, CA), and images were observed by confocal microscopy (Olympus Fluoview FV3000, Shinjuku, Tokyo, Japan) and processed using Adobe Photoshop (San Jose, CA).
Alexa 488 or 568
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488 and Alexa Fluor 568 are fluorescent dyes commonly used in biological research. They are synthetic dyes derived from the structure of the natural dye fluorescein. Alexa Fluor 488 and 568 dyes exhibit high brightness, photostability, and pH-insensitivity, making them suitable for a variety of fluorescence-based applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Phospho histone 3, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Fluoview fv3000, by Olympus (1 mentions)
Anti wt1, by Santa Cruz Biotechnology (1 mentions)
Anti six2, by Proteintech (1 mentions)
Bluemap kit, by Roche (1 mentions)
Anti wt1, by Abcam (1 mentions)
Automated discovery system, by Roche (1 mentions)
Paraformaldehyde solution, by Fujifilm (1 mentions)
Anti e cadherin, by BD (1 mentions)
Tri methyl histone h3 lys27 c36b11, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
γ tubulin, by Merck Group (1 mentions)
γh2ax, by Merck Group (1 mentions)
Horse serum, by Vector Laboratories (1 mentions)
12 protocols using alexa 488 or 568
1
Immunohistochemical Analysis of Drosophila Gut
2
Immunohistochemical Profiling of Kidney Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kidney tissues were fixed in a 4% paraformaldehyde solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), embedded in paraffin, sectioned, and stained with haematoxylin-eosin using standard methods. Immunostaining was carried out using a BlueMap Kit and automated Discovery System (Roche) or manually for immunofluorescence staining.
The following primary antibodies were used: anti-SALL1 (Perseus Proteomics, Tokyo, Japan), anti-WT1 (Santa Cruz Biotechnology), anti-WT1 (Abcam), anti-cytokeratin (Sigma-Aldrich), anti-E-cadherin (BD Sciences), anti-SIX2 (Proteintech, Rosemont, IL), and anti-SALL1 (Abcam), which recognizes the C-terminus of SALL1. Secondary antibodies were conjugated with Alexa 488 or 568 (Thermo Fisher Scientific), and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using an LSM780 confocal microscope (Zeiss, Oberkochen, Germany).
The following primary antibodies were used: anti-SALL1 (Perseus Proteomics, Tokyo, Japan), anti-WT1 (Santa Cruz Biotechnology), anti-WT1 (Abcam), anti-cytokeratin (Sigma-Aldrich), anti-E-cadherin (BD Sciences), anti-SIX2 (Proteintech, Rosemont, IL), and anti-SALL1 (Abcam), which recognizes the C-terminus of SALL1. Secondary antibodies were conjugated with Alexa 488 or 568 (Thermo Fisher Scientific), and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using an LSM780 confocal microscope (Zeiss, Oberkochen, Germany).
3
Indirect Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Indirect immunofluorescence staining was performed as described in previous studies (Oppel et al., 2019 (link), 2011 (link); Ball et al., 2017 (link)). Primary antibodies used were pan-cytokeratin (AE1/AE3; Novus Biologicals #NBP2-29429; 1:200) and tri-methyl-Histone H3 (Lys27) (C36B11; Cell Signaling Technology #9733; 1:400). Secondary antibodies used were goat anti-rabbit-IgG and goat anti-mouse-IgG conjugated with Alexa 488 or 568 (Life Technologies; 1:400).
4
Quantification of DNA Damage Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed in 4% paraformaldehyde for 30 min at room temperature. Permeabilization was carried out in 0.2% Triton X-100 in phosphate-buffered saline for 10 min at room temperature. After 1 h of saturation in 2% bovine serum albumin, immunostaining was performed using γH2AX (Millipore, Burlington, MA, USA), 53BP1 (Cell Signaling, Danvers, MA, USA), and γ-tubulin (Millipore). These antibodies were revealed via secondary antibodies coupled to Alexa 488 or 568 (Life Technologies). Nuclear staining was performed by adding 1/2000 DAPI (1 mg/mL, Life Technologies) in phosphate-buffered saline for 10 min. Slides were then analyzed with a Zeiss LSM710 confocal laser scanning microscope (60× magnification). Images were collected in the Z direction at 0.80 mm intervals and quantified using the ImageJ plug-in as described previously [14 (link)].
5
Immunohistochemical Analysis of Tau Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sagittal (5 µM) brain slices were deparaffinized and unmasked using citrate buffer (3.75 mM acid citrate, 2.5 mM disodium phosphate, pH 6) for 10 min in a domestic microwave. The slices were submerged for 1 h in 1% horse serum (Vector Laboratories), and the primary antibodies were incubated overnight at 4°C in the presence of PBS-0.2% Triton using the following primary antibodies: total Tau (Tau CTer) and Tau1 antibodies (Galas et al., 2006 (link)) and the phospho-histone H2A.X (Ser 139) antibody from Millipore.
These antibodies were revealed via secondary antibodies coupled to Alexa 488 or 568 (Life Technologies). The sections were counterstained and mounted with Vectashield/DAPI (Vector Laboratories). 4′,6-diamidino-2-phenylindole (DAPI) was used as a chromatin counterstain.
These antibodies were revealed via secondary antibodies coupled to Alexa 488 or 568 (Life Technologies). The sections were counterstained and mounted with Vectashield/DAPI (Vector Laboratories). 4′,6-diamidino-2-phenylindole (DAPI) was used as a chromatin counterstain.
6
Drosophila Ovary Immunostaining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Drosophila ovaries were dissected and immunostained as described previously (Yang et al., 2015 (link)). Briefly, ovaries were fixed in 4% paraformaldehyde for 15 min, and blocked in 5% normal goat serum in PBT (10 mM NaH2PO4, 175 mM NaCl, pH 7.4, 0.1% Triton X-100). The following primary antibodies were used: rabbit anti-pMad (1:1000, gift from Ed Laufer, Columbia University Medical Center, New York), rabbit anti-β-galactosidase (1:3000; MP Biologicals, 0855976), mouse anti-α-Spectrin (1:50; DSHB), rabbit anti-GFP (1:1000; Life, A11122), mouse anti-Flag (1:300; Sigma, F1804). Secondary antibodies, including goat anti-rabbit, anti-mouse IgGs, conjugated to Alexa (488 or 568) (Molecular Probes) were used at a dilution of 1:300 and tissues were also stained with 0.1 mg/ml DAPI (4’,6’-diamidino-2-phenylindole; Sigma) for 5 min. Images were collected using either a Zeiss LSM510/LSM 800 or Nikon A1 confocal microscope system. All acquired images were processed in Adobe Photoshop and Illustrator.
7
Immunostaining of Cardiac Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heart tissue was embedded in tissue freezing media (Triangle Biomedical Science) snap-frozen in liquid nitrogen and sectioned in the coronal plane using Leica CM1900 cryostat (Leica Microsystems, Bannockburn, IL, USA). Coronal and axial tissue sections (5 to 7 μm thickness) were mounted on positively charged glass slides and fixed/permeabilized in a 1:1 mixture of acetone and ethanol. Sections were blocked with 3% BSA in PBS and incubated with primary antibodies against α-MHC (Abcam), HDAC1, HDAC2, α-SMA (Abcam), CD90 (BD), and Vimentin (Abcam). Specific staining was visualized using corresponding secondary antibodies conjugated with Alexa 488 or 568 (Molecular Probes). Nuclei were stained with DAPI, 4′ 6-diamidino-2-phenylindole (Life Technologies).
Cells were fixed/permeabilized in a 1:1 mixture of acetone and ethanol. Cells were blocked with 3% BSA in PBS and stained with primary antibodies. Corresponding secondary antibodies were conjugated with Alexa-488 or Alexa 568 (Molecular Probes). Nuclei were stained with DAPI, 4′ 6-diamidino-2-phenylindole (Life Technologies).
Fluorescent images were captured using Leica TCS SPE confocal system configured with Leica DM 2500 microscope. Excitation maximums of 488 nm, 532 nm, and 405 nm, were used for image acquisition. Images were processed using LAS AF software (Leica Microsystems).
Cells were fixed/permeabilized in a 1:1 mixture of acetone and ethanol. Cells were blocked with 3% BSA in PBS and stained with primary antibodies. Corresponding secondary antibodies were conjugated with Alexa-488 or Alexa 568 (Molecular Probes). Nuclei were stained with DAPI, 4′ 6-diamidino-2-phenylindole (Life Technologies).
Fluorescent images were captured using Leica TCS SPE confocal system configured with Leica DM 2500 microscope. Excitation maximums of 488 nm, 532 nm, and 405 nm, were used for image acquisition. Images were processed using LAS AF software (Leica Microsystems).
8
Ovary Development Imaging Protocol
9
Immunostaining of Drosophila Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dechorionated embryos were fixed at the interface of heptane and either 18.5% paraformaldehyde (Electron Microscopy Sciences) (Postner and Wieschaus, 1994 (link)) for 30 min for actin cap staining or 4% paraformaldehyde for 70 min for mito-tagged embryo staining, in 0.1 M sodium phosphate buffer (pH 7.4). The embryos were manually devitellinized and stained with rabbit anti-GFP (1:1000, Invitrogen) and/or anti-dsRed (1:500, Invitrogen). Alexa 546 or 647-phalloidin (1:200, Invitrogen), or secondary antibodies conjugated with Alexa 488 or 568 (Invitrogen), were used at 1:500. Coronin peptide antibody was used at 1:100 dilution. Coronin peptide antibody was generated by GenScript as peptide-KLH conjugation in New Zealand rabbits (sequence in Table 3 ). The affinity-purified antibody was used at 1:100 dilution (~10 μg/mL). Embryos were mounted in ProLong Gold (Life Technologies). Olympus Fluoview FV3000 confocal laser scanning microscopy was used for immunostained embryos imaging. Exposure settings of 8 or 12 ms/pixel were used for image acquisition.
10
Immunostaining of Differentiated Cells
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!