To label Schwann cell nuclei, after treatment with ronidazole for 2 days Tg(gSAIzGFFD37A; UAS:NTR-RFP; UAS:EGFP) or Tg(gSAIzGFFD37A; UAS:NTR-RFP; zCrestHSP70:GFP) animals were fixed for 1 hour at room temperature in sweet fix (4% paraformaldehyde with 125mM sucrose in PBS) plus 0.1% Triton X-100 (Fisher, BP151). Animals were washed in phosphate buffer and incubated overnight at 4°C in primary chicken anti-GFP antibody (1:2000, Aves labs, GFP-1010) in incubation buffer (2 mg/mL BSA, 0.5% Triton X-100, 1% NGS). After washing in phosphate buffer, animals were incubated overnight at 4°C in Alexa Fluor 488 donkey anti-chicken secondary antibody (1:1000, Jackson ImmunoResearch, 703–545-155) in incubation buffer. After washing with phosphate buffer saline, larvae were incubated overnight at 4°C in Vectashield plus with DAPI (Vector Laboratories, H2000). Animals were mounted in agarose in a glass-bottomed dish and imaged in 1.5 μm slices using a x40 water immersion lens on a Zeiss LSM880 confocal microscope using Zen software. These samples were used to count total Schwann cell numbers.
Vectashield plus
Manufactured by Vector Laboratories
Sourced in United States
Vectashield plus is a mounting medium designed for fluorescence microscopy. It is formulated to maintain the brightness and stability of fluorescent signals in fixed and permeabilized samples.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 donkey anti chicken secondary antibody, by Jackson ImmunoResearch (1 mentions)
Bp151, by Thermo Fisher Scientific (1 mentions)
Gfp 1010, by Aves Labs (1 mentions)
Bz x800, by Keyence (1 mentions)
Hypoxanthine, by Thermo Fisher Scientific (1 mentions)
Agarose, by Merck Group (1 mentions)
Hoechst 33342 trihydrochloride trihydrate, by Thermo Fisher Scientific (1 mentions)
Dl lactic acid, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
N 3 dimethylaminopropyl n ethylcarbodiimide, by Merck Group (1 mentions)
Rabbit anti histamine, by Immunostar (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
1 octen 3 ol, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Electrode gel, by Parker Laboratories (1 mentions)
Opal 570, by Akoya Biosciences (1 mentions)
7 protocols using vectashield plus
1
Schwann Cell Nuclei Labeling Protocol
2
FISH Assay for c-Myc Amplification in RCC
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FISH was performed on 4 μm-thick FFPE tissue sections using c-Myc (Cy3)/Ch7CEN (Spectrum Green) dual color FISH probe (Chromosome Science Laboratory, Sapporo, Japan) as described [32 (link)] with modification. Ten additional RCC cases other than those used for aCGH analysis were randomly selected for wild-type (n = 5) and MUT (n = 5; Tables S2 and S3 ). Briefly, the slides are pretreated by boiling in 10 mM citric acid buffer (pH 6.0) with microwave for 15 min, followed by treatment with 0.5% pepsin in 0.2 N HCl at 37 °C for 30 min and washing with PBS. The slides were co-denatured with the FISH probe at 80 °C for 10 min and hybridized at 37 °C for 48 h. The slides were washed in 0.4 x saline sodium citrate (SSC)/0.3% Nonidet P-40 at 73 °C for 3 min, followed by 3 times wash with 1 x SSC at room temperature (RT). The slides were thereafter counterstained with Hoechst33342 (Thermo Fisher Scientific) and coverslips were mounted using Vectashield plus (Vector laboratories, Burlingame, CA). The samples were observed with BZ-X800 (Keyence, Osaka, Japan). The amplification was defined as c-Myc/Ch7CEN ratio of ≥2 at least in 20 nuclei per RCC cells. The average of c-Myc/Ch7CEN ratio was calculated in ≥20 cells per sample.
3
Histamine Interaction with Red Blood Cells
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Histamine (2-(1H-imidazol-5-yl)ethanamine; dihydrochloride, ACROS Organics, Fair Lawn, NJ, USA), human red blood cells (RBCs, Interstate Blood Bank, Memphis, TN, USA), and serum (Interstate Blood Bank, Memphis, TN, USA), RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with HEPES, L-glutamine, hypoxanthine (Acros Organics), DL-lactic acid (Acros Organics), Hoechst 33342-trihydrochloride trihydrate (Invitrogen, Carlsbad, CA, USA), JC-1 (Invitrogen, Carlsbad, CA, USA), mercurochrome, Ringer’s solution (3 mM CaCl2, 182 mM KCl, 46 mM NaCl, 10 mM Tris pH 7.2), 1-octen-3-ol (Sigma-Aldrich), and electrode gel (Parker Laboratories, Fairfield, NJ, USA). N-3-dimethylaminopropyl-N′-ethylcarbodiimide (Sigma-Aldrich, St. Louis, MO, USA, cat. #03449), paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA, cat. #15710), agarose (Sigma-Aldrich), goat serum (BSA; Jackson ImmunoResearch Laboratories, West Grove, PA, USA, cat. #001-000-162), rabbit anti-histamine (ImmunoStar, Hudson, WI, USA, cat. # 22939, RRID:AB_572245), Alexa Fluor 488 (Thermo Fisher, cat. #A-11008), and Vectashield®PLUS (Vector Laboratories, Burlingame, CA, cat. #H-1900).
4
Multimodal RNA in situ Analysis of Murine Nasal Tissue
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RNA in situ hybridization was performed according to manufacturer’s instructions for the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics ACD, 323270) on 20 μm thin sections of fixed-frozen murine nasal mucosa tissue collected 14 dpi. We implemented the following modifications to preserve tissue integrity: 1) 5 min PBS wash preceding initial baking of slides was removed; 2) slides were baked for 30 min at 60°C following EtOH dehydration; 3) target retrieval time was reduced to 5 min; 4) slides were baked for 60 min at 60°C following target retrieval; and 5) tissue sections were incubated in Protease Plus instead of Protease III for milder protease digestion. Probes used included Mm-Krt13 (ACD, 575341), Mm-Cxcr6-C2 (ACD, 871991-C2), Mm-Cxcl16-C3 (ACD, 466681-C3), and Mm-Cd274-C3 (ACD, 420501-C3). Following signal amplification, Opal 520 (Akoya Biosciences, FP1487001KT), Opal 570 (Akoya Biosciences, FP1488001KT), and Opal 690 (Akoya Biosciences, FP1497001KT) dyes were used, diluted 1:1000 in TSA buffer (ACD, 322809). Nuclei were stained with DAPI and slides were mounted with VECTASHIELD PLUS (Vector Laboratories, H-1900). Confocal images were collected using an Olympus FLUOVIEW FV3000 confocal laser scanning microscope.
5
Quantifying Zika Virus Infection in Mosquito Midguts
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Dissected midguts were cut in half in 1× PBS and washed at least twice in 1× PBS or until the blood bolus was completely removed. Blood-removed midguts were then fixed with 4% paraformaldehyde in PBS for 2 hours at room temperature and permeabilized using 2% Triton X-100 in 1× PBS for 15 minutes. After permeabilization, midguts were incubated in 2% bovine serum albumin in 1× PBS for 1 hour. Midguts were incubated with primary antibody anti-flavivirus envelope 4G2 antibody produced in-house at 4°C overnight followed by 1:500 secondary antibody anti-mouse IgG Alexa 488 (Invitrogen, A28175) in 1× PBS, and Hoechst33342 (Invitrogen, 62249) for nuclei staining for 3 hours at room temperature. Midguts were mounted onto glass slides in Vectashield Plus (Vector Laboratories, H1900). Images were taken at the same fluorescent settings for both ZIKV strains under an inverted fluorescence microscope (Olympus IX81) and laser scanning confocal microscopes (Nikon AXR and Carl Zeiss LSM900) using 20× objective lens (CFI Apochromat LWD Lambda S 20XC WI, LD Plan-Neofluar 20X/0.4 Corr M27). The z-section distance was 2.5 µm and the total z-section thickness was set between 30 and 60 µm. Z-section projection with maximum intensity was done using Fiji version 1.53t (8 (link)). The whole midgut tissues were used for cell-to-cell infection kinetics analyses.
6
Immunostaining of Mouse Organ of Corti
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Mice were anesthetized with intraperitoneal applications of phenobarbital (585 mg/kg) and phenytoin sodium (75 mg/kg) (Beuthanasia-D Special, Schering-Plough Animal Health, Union, NJ, USA) and sacrificed via intracardiac perfusion with 4% paraformaldehyde in PBS. The temporal bones were removed and trimmed. The staples was removed, and the round window was opened with a needle. The temporal bones were postfixed overnight in 4% paraformaldehyde in PBS at 4°C. After rinsing in PBS three times for 30 min, the temporal bones were decalcified in 10% EDTA for 48 h. The decalcified otic capsule was carefully removed, and the organ of Corti was dissected away from the modiolus. The tissue was then incubated in PBS + 0.1% Triton X-100 for 10 min, followed by staining with phalloidin-FITC (1:80, Abcam, catalog no. ab235137) in PBS + 0.1% Triton X-100 for 20 min at room temperature. The tissue was washed three times for 10 min in PBS and mounted in Vectashield Plus mounting medium (Vector Labs, Newark, CA, USA).
7
Immunocytochemistry of Epithelial Junctions
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Cells on Transwells or chips were fixed in periodate-lysine-1% paraformaldehyde 28 for 20 min at room temperature. After washing with PBS and blocking with 50 mM NH4Cl prepared in PBS, cells were permeabilized with Triton X-100 (0.1%) in PBS, and blocked with PBS containing 10% donkey serum. Primary antibodies diluted in blocking buffer were added. After incubation, cells were washed with PBS containing 2% donkey serum and secondary antibodies were added. After incubation and washes, Transwell filter membranes were excised. Membranes or chips were mounted under coverslips using Vectashield plus (Vector Labs). Antibodies used were monoclonal mouse anti-E cadherin clone M168 (Abnova), monoclonal rat anti-occludin clone 6B8A3, monoclonal rat anti-ZO-1 clone, and donkey anti-mouse or anti-rabbit Ig conjugated to Alexafluor 647 (Jackson Immunoresearch). For some cells, antibodies were omitted and either Alexafluor 647 phalloidin (Life Technologies) or CF640R wheat germ agglutinin (Biotium). Hoechst 33342 (0.5 µg/ml) was routinely included with secondary antibodies in order to detect nuclei. EGFP-claudin-2 and mCherry-ZO-1 were detected based on the fluoroprotein tag. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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