Zebrafish were euthanized in 0.08% tricaine and whole eyes were removed and fixed in 9:1 ethanolic formaldehyde (PCNA staining) or 4% paraformaldehyde (all other staining) overnight. Eyes were then washed in PBS and cryoprotected in 30% sucrose for 4 hr at room temperature. Eyes were transferred to a solution containing two parts optimal cutting temperature compound (OCT) and one part 30% sucrose overnight followed by transfer to 100% OCT for 2 hr and then embedded in OCT for cryosectioning. Antibodies used were PCNA (Sigma, P8825; Abcam, ab2426), GS (Millipore, mab302), GABA (Sigma, A0310), GABAA receptor γ2 subunit (Novus Biologicals, NB300-151), GAD65 + GAD67 (Abcam, ab11070), GFP (Torrey Pines BioLabs, TP401), and mCherry (Novus Biologicals, NBP1-96752). TUNEL labeling was performed following immunohistochemistry. The in situ cell death detection kit, TMR Red (Roche Applied Sciences, 12156792910) was used to detect apoptotic cells.
Nbp1 96752
Manufactured by Novus Biologicals
NBP1-96752 is a laboratory equipment item produced by Novus Biologicals. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
A11122, by Thermo Fisher Scientific (2 mentions)
Las 3000 luminescent image analyzer, by Fujifilm (2 mentions)
P8825, by Abcam (1 mentions)
Mab302, by Merck Group (1 mentions)
Tmr red, by Roche (1 mentions)
Ab28481, by Abcam (1 mentions)
Ab108921, by Abcam (1 mentions)
Ab6313, by Abcam (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
Shrna constructs, by Qiagen (1 mentions)
Nbd cholesterol, by Merck Group (1 mentions)
Ab25630, by Abcam (1 mentions)
Filipin, by Merck Group (1 mentions)
Filipin f9765, by Merck Group (1 mentions)
Taxol t7402, by Merck Group (1 mentions)
Nocodazole m1404, by Merck Group (1 mentions)
T6199, by Merck Group (1 mentions)
Sc 9996, by Santa Cruz Biotechnology (1 mentions)
Ab167453, by Abcam (1 mentions)
I 1000, by Vector Laboratories (1 mentions)
7 protocols using nbp1 96752
1
Zebrafish Eye Immunohistochemistry Protocol
2
Cholesterol Trafficking and Lysosomal Analysis
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Chemical and standard reagents including filipin and NBD-cholesterol were obtained from Sigma-Aldrich, St. Louis, MO. LysoTracker and fluorescent dextran were obtained from Life Technologies (Grand Island, NY). shRNA constructs were obtained from SA Biosciences (Valencia, CA). Antibodies against tubulin [1:100 for immunofluorescence (IF), T6199, Sigma-Aldrich], EGFP (1:100 for IF, 632380, Clontech) and mCherry (1:100 for IF, NBP1-96752, Novus Biologicals) have been described previously (Whyte et al., 2008 (link); Towns et al., 2009 (link)). Antibodies against StARD9 [1:100 for western blotting (WB), HPA014562] were obtained from Sigma-Aldrich. Site-directed mutagenesis utilized the Phusion kit (New England Biolabs, Ipswich, MA). Antibodies against NPC1 (5 μg/ml for IF, 1 μg/ml for WB, ab108921), LAMP1 (1:10 for IF, ab25630), SREBP1 (1:100 for IF, ab28481) and cathepsin B (1 μg/ml for IF, ab6313) were obtained from Abcam (Boston, MA). filipin (f9765) was obtained from Sigma-Aldrich. NBD-cholesterol (N1148) was obtained from Invitrogen (Waltham, MA). BacMAM-ER (C10590) was obtained from Thermo Fisher Scientific (Waltham, MA). Nocodazole (M1404) and taxol (T7402) were obtained from Sigma-Aldrich. The sequences of oligonucleotides used in this study are provided in Table S3 .
3
Western Blot Analysis of Synchronized Worms
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Synchronized worms at corresponding stages of adulthood were harvested in M9. After three rounds of freezing and thaw, worms were lysed by adding 4x loading buffer. Proteins were separated by reducing SDS-PAGE and transferred to PVDF membranes. Membranes were then blotted with primary antibodies against GFP (1:200 dilution, Cat# sc-9996, Santa Cruz), FLAG (1:2000 dilution, Cat# F7425, Sigma-Aldrich), α-tubulin (1:2000 dilution, Cat# T5168, Sigma-Aldrich), or mCherry (1:2,000 dilution, Cat# NBP1-96752, Novus Biologicals), and secondary antibodies against Rabbit IgG (1:5000 dilution, Cat# G-21234, Thermo Fisher Scientific) or Mouse IgG (1:5000 dilution, Cat# G-21040, Thermo Fisher Scientific). To harvest worms at day 5 and day 7 of adulthood, each sample was prepared from about 120 synchronized L4 worms on six 60 mm plates. The worms were transferred to fresh plates every two days until reaching desired ages. Worms undergoing internal hatching and vulva bursting were discarded. Signals of western blotting were measured using Adobe Photoshop. Background signals were subtracted as reported24 (link). All the uncropped and unprocessed images were shown in the Supplementary Fig. 8 .
4
ChIP-qPCR Assay for YY1 Binding
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The ChIP assay was performed using Pierce™ Magnetic ChIP Kit (26157, Thermo Fisher Scientific). The experimental procedures were carried out following the manufacturer’s instructions. Four micrograms of anti-YY1 (22156-1-AP, Proteintech) or anti-mCherry (ab167453, Abcam) antibody was used for the IP, while the same amount of normal rabbit IgG (I-1000, Vector Laboratories, Inc.) was used as a negative control. The anti-YY1 (1:1000, MAB3784, R&D Systems, Inc.) and anti-mCherry (1:1000, NBP1-96752, Novus Biologicals) antibodies were used for the detection of the endogenous YY1 protein and YY1-mCherry proteins in the input and IP samples. Twenty nanograms of recovered genomic DNAs from each input, normal rabbit IgG, and YY1 or mCherry immunoprecipitated samples were used in the following qPCR to analyse the levels of Fuzzy promoter fragments. The primers used were FuzzyYY1-a-F, 5′-GGTGGGTGGATCATTTGAGC-3′; FuzzyYY1-a-R, 5′-TTTTGGGAGAGGGGACAGG-3′; FuzzyYY1-b-F, 5′-GACCACAGGCCGAGTTACTC-3′ and FuzzyYY1-b-R, 5′-TTCGGTTTAGATACAGGCGTC-3′. Relative gene expression in IgG IP samples was quantified using the 2−ΔCt method, where ΔCt = CtIgG IP – CtInput. Relative gene expression in antibody IP samples was quantified using the 2−ΔΔCt method, where ΔΔCt = (Ctantibody IP – CtInput)disease – (Ctantibody IP – CtInput)control.
5
Western Blot Analysis of Cytoskeletal Proteins
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Western blots were performed according to standard procedures. The following primary antibodies were used: mouse anti-β-tubulin (1:1000, E7) and mouse anti-α-actin (1:1000, JLA20) from Developmental Studies Hybridoma Bank; and rabbit anti-Mask (1:2000) (Smith et al., 2002 (link)), mouse anti-mCherry antibody (1:1000, 1C51, NBP1-96752 Novus Biologicals), guinea pig anti-Rae1 antibody (1:500) and rabbit anti-GFP (1:1000, A11122, Invitrogen). All secondary antibodies were used at 1:10,000. Data were collected using an LAS-3000 Luminescent Image Analyzer (Fujifilm) and quantified using Multi Gauge software (Fujifilm).
6
Immunofluorescence Staining of Germ Cells
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Cells were attached to a hydrophilic micro slide glass (Matsunami Glass) and adapted to immunofluorescence staining following methods described previously (Chen et al., 2019 (link)). Cells were labeled with anti-DAZL (Abcam, AB215718; 1:200), anti-CVH (anti-DDX4; Bioss, BS-3597R; 1:200), anti-NANOG (a kind gift from Dr Kiyokazu Agata; 1:1000; Nakanoh et al., 2015 (link)) and anti-mCherry (Novus Biologicals, NBP1-96752; 1:500) antibodies and then subsequently stained with Alexa Fluor™ 488-conjugated donkey anti-rabbit IgG (Invitrogen, A-21206; 1:2000) and Alexa Fluor™ 555-conjugated donkey anti-mouse IgG (Invitrogen, A-31570; 1:2000) for visualization. Hoechst 33342 (2.5 µg/ml; Molecular Probes) was used to stain the nucleus. Images were acquired using a Leica DMI4000 B microscope (Leica Microsystems) equipped with a digital CMOS camera (Hamamatsu Corporation).
7
Western Blot Quantification Methodology
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Western blots were performed according to standard procedures. The following primary antibodies were used: mouse anti-β-Tubulin (1:1,000, E7) mouse anti-Alpha-Actin (1:1000, JLA20 ) from Developmental Studies Hybridoma Bank; rabbit anti-Mask (1:2,000) (25) , mouse anti-mCherry antibody (1:1000, 1C51, NBP1-96752 Novus Biologicals), and rabbit anti-GFP (1:1000, A11122, Invitrogen). All secondary antibodies were used at 1:10,000. Data were collected using Luminescent Image Analyzer LAS-3000 (FUJIFILM) and quantified using Multi Gauge (FUJIFILM).
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