Histological studies were performed as previously detailed by our group (Fernandes et al., 2010 (link), Laflamme et al., 2007 (link)). For immunohistochemistry, we used the primary antibodies detailed in Table S2 , followed by either fluorescent secondary antibodies (Alexa-conjugated, species-specific antibodies from Molecular Probes) or the avidin-biotin reaction followed by chromogenic detection (ABC kits from Vector Labs). In situ hybridization against the human-specific pan-centromeric probe was performed using methods previously detailed (Laflamme et al., 2007 (link)). For detection, we used a peroxidase-conjugated anti-digoxigenin antibody (Roche), followed by staining with either a chromogenic substrate or fluorescent tyramide signal amplification kit (Molecular Probes).
Peroxidase conjugated anti digoxigenin antibody
Manufactured by Roche
Sourced in United States
The Peroxidase-conjugated anti-digoxigenin antibody is a lab equipment product that functions as a specific binding reagent. It is designed to detect and bind to the organic compound digoxigenin, which is commonly used as a labeling tag in various molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Abc kit, by Vector Laboratories (1 mentions)
Leica tcs sp5 2 microscope, by Leica Microsystems (1 mentions)
Tsa plus cy5, by PerkinElmer (1 mentions)
Alkaline phosphatase conjugated anti fluorescein antibody, by Roche (1 mentions)
Peroxidase substrate, by Merck Group (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Alexa fluor 555 labeled donkey anti rabbit igg, by Beyotime (1 mentions)
Cat no 14642 1 ap, by Proteintech (1 mentions)
Cat no 15073 1 ap, by Proteintech (1 mentions)
80i microscope, by Nikon (1 mentions)
Tyramide signal amplification kit, by PerkinElmer (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Anti iba1 antibody, by Fujifilm (1 mentions)
6 protocols using peroxidase conjugated anti digoxigenin antibody
1
Histological and Immunostaining Protocols for Cell Analysis
2
In situ Hybridization of Dorsal Root Ganglia
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For in situ hybridization (ISH), mice were euthanized with
CO2. Lumbar L4–L6 DRGs were dissected and immediately frozen in
OCT on dry ice. Tissue was cryosectioned (10–12 μm), mounted onto
Superfrost Plus slides (VWR, Radnor, PA), frozen at −80°C. Digoxigenin-
and fluorescein-labeled anti-sense cRNA probes matching coding (Gprc5b, Lpar3,
TdTomato, Ntrk2 [Trkb], Prkcq, Nppb, Il31ra) or untranslated regions were
synthesized, hybridized to sections, and visualized as previously described (Liberles and Buck, 2006 (link)), with minor
modifications in amplification strategy. Following overnight hybridization, slides
were incubated with peroxidase conjugated anti-digoxigenin antibody (Roche Applied
Sciences, Indianapolis, IN, USA; 1:200) and alkaline phosphatase conjugated
anti-fluorescein antibody (Roche Applied Sciences, 1:200) for 1 hr at room
temperature. Tissues were washed and incubated in TSA-PLUS-Cy5 (Perkin Elmer)
followed by HNPP (Roche Applied Sciences) according to manufacturer's instructions.
Epifluorescence images were captured with a Leica TCS SP5 II microscope (Leica
microsystems, Buffalo Grove, IL). Sequences of primers used for probe generation are
listed inTable 3 .
CO2. Lumbar L4–L6 DRGs were dissected and immediately frozen in
OCT on dry ice. Tissue was cryosectioned (10–12 μm), mounted onto
Superfrost Plus slides (VWR, Radnor, PA), frozen at −80°C. Digoxigenin-
and fluorescein-labeled anti-sense cRNA probes matching coding (Gprc5b, Lpar3,
TdTomato, Ntrk2 [Trkb], Prkcq, Nppb, Il31ra) or untranslated regions were
synthesized, hybridized to sections, and visualized as previously described (Liberles and Buck, 2006 (link)), with minor
modifications in amplification strategy. Following overnight hybridization, slides
were incubated with peroxidase conjugated anti-digoxigenin antibody (Roche Applied
Sciences, Indianapolis, IN, USA; 1:200) and alkaline phosphatase conjugated
anti-fluorescein antibody (Roche Applied Sciences, 1:200) for 1 hr at room
temperature. Tissues were washed and incubated in TSA-PLUS-Cy5 (Perkin Elmer)
followed by HNPP (Roche Applied Sciences) according to manufacturer's instructions.
Epifluorescence images were captured with a Leica TCS SP5 II microscope (Leica
microsystems, Buffalo Grove, IL). Sequences of primers used for probe generation are
listed in
3
TUNEL Assay for Apoptosis Detection
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The specimen sections were analyzed for apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. TUNEL activity was measured using in situ cell death detection kits (Roche Diagnostic, Penzberg, Germany), according to the manufacturer’s instructions, to identify apoptotic cells in the tissue [32 (link)]. The specimens were incubated with peroxidase-conjugated anti-digoxigenin antibody (Roche Diagnostics, Penzberg, Germany). Staining was performed, and a peroxidase substrate (Sigma-Aldrich, Saint Louis, MO, USA) was used to present the color of the TUNEL reaction.
4
FISH and Immunofluorescence Imaging Protocol
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Digoxigenin-labeled RNA probes were purchased from Gene Pharma (Shanghai, China). The FISH assay was performed following a previously published protocol [39 (link)]. Briefly, the cells were fixed in 4% paraformaldehyde, permeabilized in a 1:1 solution of acetone and methanol, and hybridized with digoxigenin-labeled MYCN sense or antisense RNA probes. After peroxidase quenching and blocking, the hybridized sections were incubated with peroxidase-conjugated anti-digoxigenin antibody (Roche, Indianapolis, IN, USA) and visualized using SuperGloTM Green Immunofluorescence Amplification Kits (Fluorescent Solutions, Augusta, GA, USA). Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.). Alexa Fluor 555 labeled donkey anti-rabbit IgG (Beyotime Biotechnology) was used for fluorescence detection. Nuclei were counter-stained with DAPI. Images were obtained based on Nikon80i microscope (Nikon, Japan).
5
In situ hybridization of activity-dependent genes
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Animals were sacrificed, their brains quickly removed and immediately frozen. The frozen brains were sectioned using a cryostat microtome into 20 μm thick sections and mounted on microscope slides. The mounted tissue was fixed in 4% paraformaldehyde, permeabilized in a 1:1 solution of acetone and methanol, and then hybridized with digoxigenin-labeled Arc or Zif268 full-length antisense RNA probes. These probes were synthesized using MAXIscript® T3 and T7 (Ambion®, Austin, TX) and AmpliScribeTM T7 (Epicentre Biotechnologies, Madison, WI, USA) RNA in vitro transcription kits. After peroxidase quenching and blocking, the hybridized tissue was incubated with peroxidase-conjugated anti-digoxigenin antibody (Roche, Indianapolis, IN, USA) and revealed using SuperGloTM Immunofluorescence Amplification Kits (Fluorescent Solutions, Augusta, GA, USA) or Tyramide Signal Amplification kits (Perkin Elmer, Waltham, MA, USA) with Cyanine 3 or Fluorescein. Nuclei were stained with SYTOX® Green (Invitrogen, Carlsbad, CA, USA), DAPI, or 7-AAD.
6
Dual-Labeling FISH and Immunohistochemistry for Mouse Brain
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Immunohistochemistry and fluorescence in situ hybridization (FISH) were performed as previously described [38, 39] (link). The Trpv4, PDGFRa, plp and gfap cRNA riboprobes were described previously [26, (link)27, (link)38] . Biotin-and digoxigenin-labeled cRNA riboprobes were prepared as previously described [39] (link). Sagittal sections of 8-week-old mouse brains were hybridized with a mixture of biotin-and digoxigenin-labeled cRNA probes in hybridization buffer. Following washing, digoxigenin and biotin were detected via a two-step method. Biotin was first detected with peroxidase-conjugated streptavidin (PerkinElmer; 1:100 dilution, 1 h) and a fluorescein isothiocyanate-tyramide signal amplification (TSA) Plus Kit (PerkinElmer).
Then, after incubation with 1.0% H2O2 for 30 min to inactivate residual peroxidase, digoxigenin was detected by labeling with a peroxidase-conjugated anti-digoxigenin antibody (Roche Diagnostics; 1:500 dilution, 1 h) and an indocarbocyanine-TSA Plus Kit (PerkinElmer). An anti-Iba1 antibody (1:500 dilution, Wako) and Alexa Flour 564-conjugated goat anti-rabbit IgG (1:2000 dilution) were used to detect Iba1 after Trpv4 FISH. Sections were examined using a fluorescence microscope (BX53, Olympus) equipped with a cooled CCD camera (DP80, Olympus).
Then, after incubation with 1.0% H2O2 for 30 min to inactivate residual peroxidase, digoxigenin was detected by labeling with a peroxidase-conjugated anti-digoxigenin antibody (Roche Diagnostics; 1:500 dilution, 1 h) and an indocarbocyanine-TSA Plus Kit (PerkinElmer). An anti-Iba1 antibody (1:500 dilution, Wako) and Alexa Flour 564-conjugated goat anti-rabbit IgG (1:2000 dilution) were used to detect Iba1 after Trpv4 FISH. Sections were examined using a fluorescence microscope (BX53, Olympus) equipped with a cooled CCD camera (DP80, Olympus).
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