Formalin-fixed, paraffin-embedded monkey TM sections were used for immunochemistry staining. After deparaffinizing and antigen retrieval, two antibodies were applied to the same specimen. A mouse monoclonal antibody of 8-OHdG, (sc-393871; Santa Cruz Biotechnology, Dallas, TX, USA) was used at 1:500 dilution, and a rabbit polyclonal antibody of fibronectin (ab23751; Abcam, Cambridge, MA, USA) was used at 1:500 dilution at 4°C overnight, respectively. Alexa-Fluor 488 donkey anti-mouse IgG and Alexa-Fluor 555 donkey anti-rabbit IgG (1/1000; Abcam) were used as secondary antibodies to detect 8-OHdG and fibronectin separately. 4′,6-Diamidino-2-Phenylindole (DAPI) was used for nuclear staining. ImageJ (http://imagej.nih.gov/ij/ ; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) was used for fluorescence intensity analysis.
Alexa fluor 555 donkey anti rabbit igg
Manufactured by Abcam
Sourced in United States
Alexa Fluor 555 donkey anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 555 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 donkey anti mouse igg, by Abcam (3 mentions)
Ab23751, by Abcam (1 mentions)
Sc 393871, by Santa Cruz Biotechnology (1 mentions)
Neomycin, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Abcam (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit igg alexa fluor 647, by Abcam (1 mentions)
Alexa fluor 555 goat anti mouse igg, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Sybr green, by Roche (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Trizol reagent, by Sangon (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Tunel apoptosis assay kit, by Beyotime (1 mentions)
Ab150105, by Abcam (1 mentions)
T6793, by Merck Group (1 mentions)
Ab150074, by Abcam (1 mentions)
Tf0808, by Matsunami (1 mentions)
5 protocols using alexa fluor 555 donkey anti rabbit igg
1
Immunofluorescent Analysis of 8-OHdG and Fibronectin
2
Investigating Apoptosis and Mitochondrial Function
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GM1 (Macklin, Shanghai, China, G873919), penicillin (CSPC, Shijiazhuang, China, h20033291), CCK-8 Kits (Beyotime, Shanghai, China, C0038), BeyoRTTM II First-Strand cDNA Synthesis Kits with gDNA Eraser (Beyotime, D7170 M), TUNEL Apoptosis Assay Kits (Beyotime, C1086), DMSO (Sigma-Aldrich, Saint Louis, MO, United States, D8371), neomycin (Sigma-Aldrich, N6386-5G), paraformaldehyde (Sigma-Aldrich, 158127), TRIzol reagent (Sangon Biotech, Shanghai, China, B610409-0100), SYBR Green (Roche, Basel, Switzerland, 4913914001), MitoSOX Red (Life Technologies, Carlsbad, CA, United States, M36008), TMRE Mitochondrial Membrane Potential Assay Kits (Abcam, Cambridge, United Kingdom, ab113852), Triton X-100 (Solarbio, Beijing, China, T8260), DAPI (Solarbio, C0060), antibodies against cleaved CASPASE-3 (Thermo Fisher Scientific, Carlsbad, CA, United States, Cat# 43-7800, RRID:AB_2533540 ), antibody against MYOSIN 7a (Santa Cruz Biotechnology, Santa Cruz, United States, Cat# sc-74516, RRID:AB_2148626 ), Alexa Fluor 488 goat anti-rabbit IgG (Abcam, Cat# ab150077, RRID:AB_2630356 ), Alexa Fluor 555 donkey anti-rabbit IgG (Abcam, Cat# ab150062, RRID:AB_2801638 ), Alexa Fluor 555 goat anti-mouse IgG (Abcam, Cat# ab150118, RRID:AB_2714033 ), and Alexa Fluor 647 donkey anti-rabbit IgG (Abcam, Cat# ab150077, RRID:AB_2630356 ) were used in this study.
3
Immunostaining of Sperm Axonemal Proteins
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Spermatozoa in Hank’s buffer were attached to the wells of an eight-well glass slide (TF0808; Matsunami). After washing out excess sperm, attached spermatozoa were briefly demembranated using 1% Nonidet P-40 for 2 min. Specimens were fixed with 2% paraformaldehyde/Hank’s buffer for 10 min at room temperature, followed by treatment with cold acetone and methanol (−20°C). After rehydration with PBST (phosphate buffered saline containing 0.1% Tween-20), specimens were treated with blocking buffer (2% normal goat serum, 1% cold fish gelatin in PBST). Immunostaining was performed with anti-acetylated tubulin antibody (1:500 dilution; T6793; Sigma-Aldrich) and anti-Dnah8 antibody (1:50 dilution; generated in this study) as primary antibodies. Alexa Fluor 488 Donkey anti-mouse IgG (1:250 dilution; ab150105; Abcam) and Alexa Fluor 555 Donkey anti-rabbit IgG (1:250 dilution; ab150074; Abcam) were used as secondary antibodies with 2.5 μg/ml DAPI (Wako) for nuclear staining. Specimens were mounted with Fluoro-KEEPER Antifade Reagent (Nacalai tesque) and observed with a fluorescence microscope (BX60; Olympus) and a CCD camera (ORCA-R2; Hamamatsu).
4
Quantifying TFEB Nuclear Translocation
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Antibodies were prepared in the following concentrations: TFEB 1:100 (Abcam), p62 1:100 (BD Biosciences, San Jose, CA, United States) and LAMP-1 1:100 (Santa Cruz Biotechnology, Dallas, TX, United States); secondary antibodies were Alexa Fluor 555 donkey anti-goat IgG 1:100 (Abcam), Alexa Fluor 647 donkey anti-goat IgG 1:100 (Thermo Fisher Scientific), Alexa Fluor 555 donkey anti-rabbit IgG (Abcam) and Alexa Fluor 488 donkey anti-mouse IgG 1:100 (Abcam). DAPI (Cell Signaling Technology) was used at a concentration of 1:10,000. Slides were viewed on a Zeiss LSM 700 confocal microscope (Carl Zeiss Canada, Toronto, ON, Canada). TFEB nuclear translocation was assessed on Adobe Photoshop (CS4) (San Jose, CA, United States) as the number of red pixels in at least three randomly selected x630 fields, in at least three experimental replicates per condition for NRK-52E cells or in kidney sections from vehicle- (n = 5) or Tubastatin A- (n = 4) treated rats.
5
Cytoskeletal Protein Localization in Cells
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After fixation with 1% paraformaldehyde (Santa Cruz Biotechnologies, Dallas, TX, USA) and permeabilization with 0.05% saponin (Sigma-Aldrich, St. Louis, MO, USA), cells were incubated with anti-alpha 1 spectrin antibodies (abs) (mouse monoclonal); anti-ankyrin erythroid/ANK abs (mouse monoclonal); anti-band 3/AE 1 abs (rabbit monoclonal) or f-actin staining kit, respectively. Secondary abs were Alexa Fluor 488 goat anti-mouse IgG or Alexa Fluor 555 donkey anti-rabbit IgG (all Abcam, Cambridge, UK). Unspecific reactions were blocked with 10% goat serum (Sigma-Aldrich, St. Louis, MO, USA). Imaging was performed on a Nikon Eclipse Ti microscope (Amstelveen, the Netherlands). The mean fluorescence intensity over the whole image field was quantified with the Nikon General Analysis 3 Software (version 1.3.660.548).
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