Slides were dewaxed in clearene and rehydrated through a graded series of ethanol to water. This was followed by 3 × 5‐minute incubations in phosphate‐buffered saline (PBS, 1X PBS). Tissue samples were circled using a wax pen and covered with permeabilizing solution (0.05% Triton X‐100 in 1X PBS, Sigma‐Aldrich, United States) and incubated for 60 minutes. This was followed by blocking with 1% BSA (Sigma‐Aldrich, United States) and PBS for 45 minutes. The primary antibody against Cx43 (Sigma C6289; Sigma‐Aldrich, United States) was applied at 1:1000 dilution and incubated overnight. Negative controls were incubated overnight in blocking solution containing no primary antibody. Slides were washed 3 × 5 minutes in PBST (0.05% Tween 20 in 1X PBS, Sigma‐Aldrich, United States) before incubation for 1 hour at room temperature with the secondary antibody (Alexa555 goat antirabbit 1:500; ThermoFisher, United States). A counterstain of DAPI (1:10 000 in PBS, ThermoFisher, United States) was applied as a nuclear marker. After a further 3 × 5 minutes PBS washes, the slides were coverslipped using Citiflour (Citifluor Ltd, London, UK) mounting medium and imaged on a confocal microscope (Leica SP8; Leica, Germany) at 40× magnification. All images were acquired using identical parameters and settings to allow for direct comparison of staining intensity.
Alexa555 goat anti rabbit
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 555 goat anti-rabbit is a secondary antibody conjugated with Alexa Fluor 555 dye, designed for use in immunoassays and other fluorescence-based applications. It is specific for rabbit primary antibodies and can be used to detect and visualize target proteins or other biomolecules labeled with a rabbit primary antibody.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Naltrexone hydrochloride, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit, by Cell Signaling Technology (1 mentions)
Cd206, by Bio-Rad (1 mentions)
Anti phospho p70s6k and anti p70s6k antibodies, by Cell Signaling Technology (1 mentions)
Ifn γ, by Genentech (1 mentions)
Fccp oligomycin, by Agilent Technologies (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Cell energy phenotype test kit, by Agilent Technologies (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Ab4412, by Abcam (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Chicken anti p0, by Aves Labs (1 mentions)
Goat anti mouse alexa 555, by Thermo Fisher Scientific (1 mentions)
Alexa555 goat anti chicken, by Thermo Fisher Scientific (1 mentions)
Mouse anti nf200, by Abcam (1 mentions)
6 protocols using alexa555 goat anti rabbit
1
Immunohistochemical Staining of Connexin 43
2
Naltrexone Effects on Macrophage Phenotypes
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Naltrexone hydrochloride (Sigma Aldrich, St. Louis, MO, USA) (−)- naltrexone stereoisomer was used for all experiments and dissolved in purified H2O as per the manufacturer’s instructions. Tested concentrations for Naltrexone hydrochloride were 50 μM, 100 μM, 250 μM, 500 μM and 1000 μM. For immunofluorescence, we used iNOS (Thermo Fisher Scientific, Waltham, MA, USA) and CD206 (BioRad, Hercules, CA, USA) monoclonal antibodies. Secondary antibodies used were Alexa 488 goat anti-mouse IgG1, Alexa 488 and 555 goat anti-rat IgG2a and Alexa 555 goat anti-rabbit secondary reagents (all from Thermo Fisher Scientific, Waltham, MA, USA). The blue-fluorescent DNA stain DAPI was used for nuclear staining (Thermo Fisher Scientific, Waltham, MA, USA). mTOR signalling was assessed using anti-phospho-p70S6K and anti-p70S6K antibodies (Cell Signalling Technologies, MA, USA), while anti β-Actin (Sigma Aldrich, St. Louis, MO, USA) was used for protein loading control. The secondary antibody for Western blot analysis was HRP conjugated goat anti-rabbit (Cell Signalling Technologies, MA, USA). Real-time metabolism assay was performed using IFN-γ (Genentech, San Francisco, CA, USA), LPS (Sigma Aldrich, St. Louis, MO, USA) and FCCP/Oligomycin as part of the Cell Energy Phenotype Test Kit (Agilent, Santa Clara, CA, USA).
3
Fetal and Adult Ovary Immunohistochemistry
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Human fetal testis tissue (9 weeks post-conception) was provided with approval from the
Human Developmental Biology Resource (HDBR,www.hdbr.org ). Term fetal (40 weeks) ovary
was obtained from Abcam (#ab4412) and adult ovary tissue (19 years) was obtained from
Tissue Solutions. Tissue sections (12 µm) were fixed briefly in 4% PFA in TBS, rinsed in
TBS and blocked in 1% BSA in TBS-Tween (0.5% Tween) for 1 h before incubating overnight
with rabbit monoclonal SOX8 antibody (Sigma HPA41640; 1: 200 dilution) and, where
relevant, mouse monoclonal NR5A1 (SF1) antibody (Thermo Fisher #434200; 1: 200
dilution). Sections were washed in TBS-Tween and incubated for 1 h with the relevant
secondary antibodies: Alexa555 goat anti-rabbit (Invitrogen, A21429; 1: 400) and
Alexa488 goat anti-mouse (Invitrogen, A11001; 1: 400). Nuclei were counterstained with
DAPI (Sigma). Slides were washed and mounted using ProLong Gold Antifade Mountant
(Lifetech, #P36930). Images were collected on a Zeiss LSM 710 confocal microscope (Carl
Zeiss) and analysed using Zeiss Zen 2009 and Image J.
Details of the plasmids, cell lines, cellular localization assays, structural modelling
and co-immunopreciptation are provided in the SI methods.
Human Developmental Biology Resource (HDBR,
was obtained from Abcam (#ab4412) and adult ovary tissue (19 years) was obtained from
Tissue Solutions. Tissue sections (12 µm) were fixed briefly in 4% PFA in TBS, rinsed in
TBS and blocked in 1% BSA in TBS-Tween (0.5% Tween) for 1 h before incubating overnight
with rabbit monoclonal SOX8 antibody (Sigma HPA41640; 1: 200 dilution) and, where
relevant, mouse monoclonal NR5A1 (SF1) antibody (Thermo Fisher #434200; 1: 200
dilution). Sections were washed in TBS-Tween and incubated for 1 h with the relevant
secondary antibodies: Alexa555 goat anti-rabbit (Invitrogen, A21429; 1: 400) and
Alexa488 goat anti-mouse (Invitrogen, A11001; 1: 400). Nuclei were counterstained with
DAPI (Sigma). Slides were washed and mounted using ProLong Gold Antifade Mountant
(Lifetech, #P36930). Images were collected on a Zeiss LSM 710 confocal microscope (Carl
Zeiss) and analysed using Zeiss Zen 2009 and Image J.
Details of the plasmids, cell lines, cellular localization assays, structural modelling
and co-immunopreciptation are provided in the SI methods.
4
Quantifying Sumoylation in Muscle Nuclei
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Tissue preparation and fluorescence staining was conducted as previously described [21 (link)]. Alexa488 goat anti-mouse (Invitrogen, Karlsruhe, Germany; dilution 1:500) and Alexa555 goat anti-rabbit (Invitrogen, Karlsruhe, Germany; dilution 1:500) secondary antibodies were used to determine type I myofibres, lamin-A and SUMO-1 respectively. Pictures were taken by a Zeiss confocal laser scanning microscope equipped with Plan-Neofluar 40× and 63×/1.3 Oil DIC objectives (LSM 510Meta, Zeiss, Jena, Germany). Alexa488 was excited by an Argon laser using the filter set BP505-530, Alexa555 by a Neon laser using the filter set BP565-615. Intranuclear SUMO-1localization was conducted by determining the fluorescence staining intensity of SUMO-1 (244 ± 54 nuclei per time-point of subjects) doubly-labeled for SUMO-1 (Alexa 555) and lamin-A (Alexa 488) in pictures with 40× fold magnification. The quantification of intranuclear SUMO-1 signal was conducted via ImageJ® by using the line scan function exclusively within the inner borders of lamin-A stained nuclear envelope of nuclei.
5
Immunocytochemistry for Neural Markers
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To check for the expression of typical SKP and SC markers, immunocytochemistry was achieved. After fixed in 4 % paraformaldehyde for 10 min, cells were blocked with 5 % normal goat serum and permeabilized with 0.1 % Triton-X for 30 min. Primary antibody was incubated overnight at 4 °C, followed by a secondary antibody incubation for 2 h at room temperature. Where Hoechst was used, 1:5000 Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) was added for incubating 5 min. The following primary antibodies were used at the stated dilutions: mouse anti-nestin antibody (1:200, Millipore, Temecula, CA, USA), rabbit anti-Sca-1 (1:200, Merck Millipore, Darmstadt, Germany), rabbit anti-versican, fibronectin, vimentin, collagen I, collagen IV (1:200, Abcam, Cambridge, MA, USA), chicken anti-P0 (1:500, Aves Labs, Davis, CA, USA), mouse anti-S100β and anti-laminin (1:500, Sigma Aldrich, St Louis, MO, USA), rabbit anti-GFAP (1:500, Dako, Tokyo, Japan), mouse anti-p75NTR (1:500, Chemicon, Temecula, CA, USA), mouse anti-NF200 (1:400, Abcam, Cambridge, MA,USA), and rabbit anti-MBP (1:500, Millipore, Billerica, MA, USA). The following secondary antibodies were used at the stated dilutions: Alexa 555 goat anti-chicken, Alexa 555 goat anti-rabbit, Alexa 488 goat anti-mouse, Alexa 555 goat anti-mouse, (all 1:1000, all Invitrogen, Carlsbad, CA, USA).
6
Measuring Proliferation Rates in Breast Cancer Cells
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5-bromo-2'-deoxyuridine (BrdU or BrdUrd) incorporation in MCF-7 and BT-474 cells was calculated by dual staining with human CK18 (rabbit polyclonal AP1021; Calbiochem, La Jolla CA) and BrdU (mouse monoclonal #347580; Becton-Dickinson, San Jose CA), followed by red Alexa-555 goat anti-rabbit and green Alexa-488 goat anti-mouse antibodies (Invitrogen). Basal MDA-MB-231 and BT-20 were stained for human CD44 (rabbit monoclonal 1998–1; Epitomics) or CK5 (rabbit monoclonal 2290–1; Epitomics) instead of CK18. For cells grown in conditioned media, BrdU quantitation was performed by immunocytochemistry (ICC) using Image J software. For 3D cultures immunohistochemistry (IHC) was used. Total cells were quantified by counterstaining with blue fluorescent 4’-6-diamidino-2-phenylindole (DAPI). Antibody against phosphorylated Histone H3 (Rabbit pAb Millipore # 06–570) was used for IHC as described [30 (link)].
Proliferation rates were calculated by the ratio of BrdU + nuclei (green) to DAPI + nuclei (blue) in CK18+, CD44+ or CK5+ cells (red) using Image Pro 4.5 software (Media Cybernetics). Quantification of BrdU incorporation and phosphorylated Histone H3 assays were performed in a minimum of five different fields from three independent experiments.
Proliferation rates were calculated by the ratio of BrdU + nuclei (green) to DAPI + nuclei (blue) in CK18+, CD44+ or CK5+ cells (red) using Image Pro 4.5 software (Media Cybernetics). Quantification of BrdU incorporation and phosphorylated Histone H3 assays were performed in a minimum of five different fields from three independent experiments.
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