Spermatocyte spreads were prepared and stained as described elsewhere (34 (link),35 (link)). To visualize stained chromosomes, Z-stacks of each channel were taken on a Zeiss AxioImager M2 microscope, using the Axiovision software package (Zeiss). SCP3 and γH2AX patterns were used to stage spermatocytes, and intensity adjusted for presentation purposes. Primary antibodies (and dilutions) used for immunoflouresence were: SCP3 (Abcam, 1:500), γH2AX (EMD Millipore, 1:1000), rabbit anti-mRad18 (described by Tateishi et al. (36 (link)), 1:100), rabbit-anti-FANCD2 (Epitomics, 1:50); Secondary antibodies, donkey-anti-rabbit-Alexa647 and donkey-anti-mouse-Alexa568 (Invitrogen) were diluted 1:500. DNA was counterstained with DAPI.
Donkey anti rabbit alexa 647
Manufactured by Thermo Fisher Scientific
Sourced in United States
Donkey anti-rabbit Alexa 647 is a secondary antibody conjugated with Alexa Fluor 647 dye. It is used to detect and visualize rabbit primary antibodies in various applications such as immunofluorescence, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Donkey anti mouse alexa 488, by Thermo Fisher Scientific (9 mentions)
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
Donkey anti rabbit alexa 488, by Thermo Fisher Scientific (3 mentions)
Donkey anti rat cy5, by Jackson ImmunoResearch (2 mentions)
Rabbit anti tmem119, by Abcam (2 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (2 mentions)
Anti perilipin 3, by Progen Biotechnik (2 mentions)
Alexa 555 donkey anti rabbit, by Thermo Fisher Scientific (2 mentions)
Donkey anti rabbit alexa 405, by Thermo Fisher Scientific (2 mentions)
Donkey anti guinea pig alexa 488, by Jackson ImmunoResearch (2 mentions)
Rabbit anti iba1, by Fujifilm (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Rat anti cd68, by Bio-Rad (2 mentions)
Alexa 594 donkey anti rat, by Thermo Fisher Scientific (2 mentions)
Alexa 488 donkey anti goat, by Thermo Fisher Scientific (2 mentions)
Donkey anti mouse alexa 647, by Thermo Fisher Scientific (2 mentions)
Neuronal nuclei (neun), by Merck Group (2 mentions)
Cytofix fixation buffer, by BD (2 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (2 mentions)
Phosflow perm buffer 3, by BD (2 mentions)
21 protocols using donkey anti rabbit alexa 647
1
Spermatocyte Spreads Immunofluorescence Staining
2
Multimodal Immunostaining of Brain Sections
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Free-floating sections were washed three times in PBS, followed by 1 hour blocking in PBS with 10% donkey serum. Sections were incubated in PBS with 10% donkey serum and primary antibodies for 48 hours at 4 °C: rabbit anti-TMEM119 (1:400, Abcam, ab209064), rat anti-CD68 (1:200, Bio-Rad, MCA1957GA), rabbit anti-Iba1 (1:1000, Wako, 019–19741), guinea pig anti-Perilipin 3 (1:200, Progen, G37). After the primary antibody incubation, sections were washed three times in PBS and incubated in PBS with 10% donkey serum and secondary antibodies for 3 hours at room temperature (RT): donkey anti-rabbit Alexa 555, donkey anti-rabbit Alexa 647, donkey anti-rabbit Alexa 405 (all 1:500, Invitrogen), donkey anti-rat Cy5, donkey anti-guinea pig Alexa 488 (all 1:500, Jackson Immuno Research). Sections were washed once in PBS and incubated in PBS with BODIPY™ 493/503 (1:1000 from 1 mg/ml stock solution in DMSO; ThermoFisher) to stain lipid droplets and Hoechst 33342 (1:2000, ThermoFisher) for nuclear counterstaining for 15 min at RT. Sections were mounted on microscope slides and embedded with Vectashield (H-1000, Vector Laboratories). Note that for successful lipid droplet staining, antigen retrieval steps and treatment with detergents have to be avoided, and sections should be embedded while still wet.
3
Immunostaining for TEAD1 Protein
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Immunostaining was performed directly in the wells. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, then washed with PBS 3 times. The cells were permeabilized with 0.1% Triton X-100 (Sigma, T8787) in PBS for 5 min, then blocked with blocking buffer [PBS supplemented with 0.5% BSA and 0.1% Triton X-100] for 1 h. Cells were incubated with the primary antibody diluted in the blocking buffer overnight at 4 °C. The following primary antibody was used: anti-TEAD1, 1:100 (Cell Signaling Technology, 12292S). The cells were washed 3 times in PBS, then incubated with the secondary antibody diluted in blocking buffer for 1 h at room temperature. The following secondary antibody was used: donkey anti-rabbit-Alexa 647, 1:500 (Invitrogen, A-31573). The nuclei were stained with Hoechst 33258 (Thermo Fisher, H3569). Cells were washed 3 times in PBS, then imaged with a Leica DMi-8 fluorescence microscope. Some images were globally adjusted for brightness and/or contrast.
4
Immunofluorescence Labeling of Spinal Cord and Brain
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Spinal cord and brain sections were rinsed in 1× tris-buffered saline (TBS), followed by incubation overnight at 4°C with primary antibodies diluted in blocking solution containing 0.5% gelatin (Bio-Rad) and 0.01% triton-X (Sigma-Aldrich) in 1×TBS. The following day, the sections were rinsed once again in 1×TBS before incubation at room temperature for 1.5 hours with the secondary antibodies. Before mounting the slides, the tissue was rinsed repeatedly in 1×TBS. The primary antibodies used were mouse monoclonal anti-NeuN 1:400 (Millipore, Solna, Sweden), chicken anti–green fluorescent protein 1:1000 (Abcam, Cambridge, United Kingdom), and rabbit anti-Pax2 1:200 (Biolegend, San Diego, CA). The secondary antibodies used were goat anti-chicken Alexa 647 1:200 (Invitrogen), donkey anti-mouse Alexa 488 1:400 (Invitrogen), and donkey anti-rabbit Alexa 647 1:400 (Invitrogen). IB4 was diluted 1:50 (conjugated to Alexa 647; Invitrogen) and 4′,6-diamidino-2-phenylindole (Sigma-life science) at 1:1000.
5
Multimodal Immunostaining of Brain Sections
6
Multicolor Immunofluorescence Staining Protocol
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IFA was performed by combining ON goat anti-CD4 (R&D system, 1:1000 cat#AF-379-NA), rabbit anti-KI67 (Thermo science, 1:200 cat# MA5-14520) and mouse anti-BCL6 (DAKO, 1:100) in TBS-tween. Slides were washed, incubated with secondary antibodies donkey anti-goat IgG-Alexa 488, donkey anti-mouse IgG-Alexa 594 and donkey anti-rabbit Alexa 647 (Molecular Probes and ThermoFisher Scientific) for 1hat room temperature. To decrease autofluorescence, the tissues were incubated with Sudan Black solution (0.1% in 80% ethanol [ENG Scientific, Inc.] + 1x TBS); for 30 min at room temperature, washed, counterstained with DAPI (RTU; ACD) for 10 min, washed in TBS and cover slipped using Prolong Gold reagent (Invitrogen). IFA images were visualized and photographed with Zeiss Axio Imager Z1 microscope (Zeiss) affixed with Apotome and total Tfh and Tfh expressing Ki67 were counted using FIJI software.
7
Immunofluorescence Staining of Drosophila Embryos
8
Immunofluorescence Staining in Diverse Tissues
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For immunofluorescence staining in skin, pancreas, mammary gland and intestine, sections were thawed at room temperature for 15 min and encircled with DAKO hydrophobic pen. Then, they were washed 3x for 5 min with PBS. Antigen retrieval was performed by adding pre-warmed citrate buffer pH=6.0 to the samples and incubating them at 85°C for 30 min. Samples were washed 3x for 5 min with PBS. Samples were incubated in blocking solution (10% horse serum, 0.5% Triton X-100 in PBS) for 1h at room temperature. Primary antibodies were diluted in staining solution (5% horse serum, 0.5% Triton X-100 in PBS) and added to the samples over night at 4°C. Next day, the samples were washed 3x for 5min with PBS and incubated with secondary antibodies (1:1000) and Hoechst (Sigma, 1mg/ml stock, 1:1000) diluted in staining solution for 2hrs at room temperature. After washing 3x for 5min with PBS, samples were mounted with Mowiol and stored at 4°C until they were imaged at a Zeiss LSM800. Primary antibodies: Keratin 8 (Abcam), Keratin 14 (BioLegend), beta-Catenin (Cell Signaling). Secondary antibody: donkey anti-rabbit Alexa647 (Molecular Probes). Mounted sections were washed 3x for 5 min in PBS, DAPI stained (1:20'000) for 10 min and then embedded in mounting medium containing 1,4diazabicyclooctane (DABCO; Roth) and Mowiol (Roth).
9
Immunohistochemical Characterization of Ca2+ Pumps
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Anesthetized mice (C57BL/6) were transcardially perfused with 0.1 phosphate buffer (PB) followed by 4% PFA (in PB) for 10 min. Brains were washed twice in PB for 1h and cut sagitally on a vibratome (80 mm). Slices were blocked and permeabilized with 5% BSA and 0.1% Triton X-100 for 1 hr at RT followed by incubation with anti-PMCA2 (ab3529; Abcam), anti-PMCA3 (sc-390148; Santa Cruz Biotechnology), anti-neuroplastin-a and anti-basigin overnight at 4 C. Immunoreactivity of the respective proteins was visualized with donkey-anti-rabbit-Alexa647, donkey-anti-mouse-Alexa488, donkey-anti-sheep-Alexa555 and donkey-anti-goat-Alexa555 secondary ABs (Molecular Probes).
10
Antibody Immunostaining in Drosophila Embryos
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The following primary antibodies were used in this study: Chicken anti-GFP IgY (1:1000, Life Technologies A10262, Carlsbad, CA, USA); Rabbit anti-Whacked peptide (1:750) [69 (link)]; rabbit anti- aPKC ζ H-300 (1:200, Santa Cruz); mouse anti-aPKC ζ A-3 (1:200, Santa Cruz); mouse anti flag M2 (1:1000, Sigma Aldrich); mouse anti- Acetylated tubulin clone 6–11 B-1(1:2000, Sigma Aldrich); Rabbit anti-Verm (1:500) [49 (link)]; mAb2A12 (1:5, DSHB, Iowa City, IA); rat anti-Trh (1:500 final dilution, this study) ; mouse anti-β-galactosidase (1:5000–1:1000; Millipore-Sigma). The following secondary antibodies (Life technologies) were used: goat anti-chicken Alexa 488, donkey anti-mouse IgG Alexa 555, goat anti-rat Alexa 555, donkey anti-rabbit Alexa 647, and donkey anti-mouse IgG Alexa 647 (1:1000 each). To visualize chitin in fixed embryos, a TMR Star-conjugated chitin-binding probe (NEB) was incubated along with secondary antibodies (1:1000).
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