Lung cells were seeded on glass coverslips and exposed to NMP after one day. NMP uptake was studied with a live-cell high-content imaging system (Operetta CLS; PerkinElmer), and algorithm-driven image quantification was performed using dedicated imaging software (Harmony 4.9). For immunofluorescence microscopy of samples, cells were fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich) for 20 min and permeabilized with Triton X-100 (0.01% in PBS; Sigma-Aldrich). Fixed cells were incubated with primary antibodies: Nrf2, β-catenin, HO-1 (all Cell Signaling), and FITC- or FR- (flash red) labeled phalloidin for actin cytoskeleton staining (both BioLegend), followed by staining with secondary antibodies conjugated to Alexa Fluor 488 for red NMP or Alex Fluor 594 for green NMP, respectively (both Life Technologies, Ober-Olm, Germany). DAPI was used for nuclear counterstaining, and samples were mounted onto glass slides (VectaShield; Biozol, Hamburg, Germany) prior to fluorescence microscopy using an Axio Observer Z.1 (Zeiss).
Vectashield
Manufactured by Calibre Scientific
Sourced in Germany
Vectashield is a mounting medium designed for use with fluorescence microscopy. It is a water-based, glycerol-based formulation that is used to mount and preserve fluorescently-labeled samples for microscopic observation.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Axio observer z1, by Zeiss (2 mentions)
Alexa 488, by Thermo Fisher Scientific (2 mentions)
Lsm880, by Calibre Scientific (2 mentions)
Paraformaldehyde pfa, by Merck Group (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Operetta cls, by PerkinElmer (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Time lapse microscopy, by Zeiss (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Lsm710 laser scanning unit, by Zeiss (1 mentions)
Plan neofluar, by Zeiss (1 mentions)
Mab1281, by Merck Group (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab28364, by Abcam (1 mentions)
Cell observer z1, by Zeiss (1 mentions)
Cryostat, by Zeiss (1 mentions)
7 protocols using vectashield
1
NMP Uptake in Lung Cells
2
Visualization of Keratinocyte Wound Healing
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One million HaCaT keratinocytes were seeded onto 60 mm plastic culture dishes and incubated overnight to permit cell adhesion. Scratches were performed using a 10 μL pipette tip. Gap closure was followed by time lapse microscopy (Zeiss, Germany) and three gap distances per samples were evaluated at different time points using Axio Vision software (Zeiss). For immunofluorescence, HaCaT keratinocytes were grown on glass coverslips for 24 h prior to fixation (4% paraformaldehyde; Sigma) and permeabilization (PBS with 0.1% Triton X-100; Sigma). Samples were subsequently incubated with primary antibodies (1 : 500; cell signaling technologies, USA) targeted at zona occludens protein 1 (ZO-1) overnight at 4°C. Cells were washed twice and incubated with appropriate Alexa Fluor 488-conjugated secondary antibodies (1 : 700; life technologies) for 1 h. Coverslips were washed and mounted onto glass microscope slides using mounting medium containing DAPI (VectaShield; Biozol, Germany) prior to analysis using an Axio Observer Z.1 (Zeiss).
3
Immunofluorescence Staining of Ubiquitin and Vimentin
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Cells were seeded and treated on coverslips. After 24 h, the coverslips were washed with PBS once and fixed in an acetone-methanol-solution (3:7) at -20 °C for 8 min. Blocking solution (10% BSA, 0.25% triton X-100 in PBS) was added and incubated for 1 h at room temperature. Incubation with the primary antibodies was carried out overnight in a wet chamber at 4 °C. Antibodies against ubiquitin (1:100) and vimentin (1:200) were diluted in blocking solution. The next day, the coverslips were washed with PBS (thrice for 5 min) and incubated for 1 h at room temperature with secondary antibodies (1:400, diluted in blocking solution). Cells were washed twice with PBS, once with high salt PBS (0.4 M NaCl in PBS) for 2 min, and once more with PBS. Using a scalpel and tweezers, we carefully placed the coverslips on slides and added 10 µL Vectashield® (Biozol, Vec-H-1.000) containing TO-PRO™-3 (Thermo Fischer, T3605) in a dilution of 1:100 to stain the nuclei. Images were captured with the confocal microscopy Zeiss Axio Observer.Z1 microscope equipped with a LSM710 laser-scanning unit (Carl Zeiss, Jena, Germany). Analysis was performed with the software ImageJ.
4
Drosophila Muscle Development Analysis
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Drosophila strains were grown under standard conditions at 27 °C to enhance GAL4 activity. All RNAi knock-downs were induced with Mef2-GAL4, a GAL4 line specifically expressed during development of all muscle types of the fly57 (link). UAS RNAi lines used were from the Vienna69 (link) and Harvard collections70 (link) and ordered from VDRC or Bloomington stock centres. See Supplementary Table S6 for all genotypes used. The flight test was done as described in57 (link).
Indirect flight muscle morphology was analysed in 3 to 5 days adult males or in 90 h APF pupae as previously published71 (link). Briefly, 90 h APF pupae or adult flies were fixed with 4% paraformaldehyde in PBT (PBS, 0.5% triton X-100) and cut into half-thoraces using a sharp microtome blade. The half-thoraces were blocked with 3% normal goat serum in PBT for 30 min and F-actin in the flight and leg muscles was visualised with phalloidin (coupled to Alexa488 or rhodamine, Molecular Probes, 1:1000 in PBT for 2 h or overnight) and Sls was stained with anti-Kettin antibody MAC155 (Babraham Institute, 1:100 in PBT overnight). The stained half-thoraces were imbedded in Vectashield (Biozol) and imaged on an LSM780 or LSM880 confocal microscope. Images were processed using Fiji72 (link).
Indirect flight muscle morphology was analysed in 3 to 5 days adult males or in 90 h APF pupae as previously published71 (link). Briefly, 90 h APF pupae or adult flies were fixed with 4% paraformaldehyde in PBT (PBS, 0.5% triton X-100) and cut into half-thoraces using a sharp microtome blade. The half-thoraces were blocked with 3% normal goat serum in PBT for 30 min and F-actin in the flight and leg muscles was visualised with phalloidin (coupled to Alexa488 or rhodamine, Molecular Probes, 1:1000 in PBT for 2 h or overnight) and Sls was stained with anti-Kettin antibody MAC155 (Babraham Institute, 1:100 in PBT overnight). The stained half-thoraces were imbedded in Vectashield (Biozol) and imaged on an LSM780 or LSM880 confocal microscope. Images were processed using Fiji72 (link).
5
CD44 Variant Expression in Prostate Cancer
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To analyze CD44v4, v5, and v7 distribution in PC3, tumor cells (treated with 5 µg/mL sE-cadherin for 72h versus non-treated) were transferred to 8-well polystyrene culture slides (Falcon®; Merck Millipore, Darmstadt, Germany). Cell cultures were then washed and fixed in cold (−20 °C) methanol/acetone (60/40 v/v). Subsequently, cells were incubated for 60 min with APC-conjugated anti-CD44v4, v5, or v7 monoclonal antibodies. To prevent photobleaching of the fluorescent dye, cells were then embedded in an antifade reagent/mounting medium mixture including DAPI (VECTASHIELD, Antifade Mounting Media, Biozol, Eching, Germany). Cells were then viewed using a confocal laser scanning microscope (LSM 10; Zeiss, Jena, Germany) with a Plan-Neofluar ×63/1.3 oil immersion objective.
6
Immunofluorescence Analysis of Tumor Samples
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Tumor samples were snap frozen in OCT and stored at -80°C until processing. Tissue blocks were sectioned into 8 μm slices on a cryostat (Zeiss, Germany). Sections were fixed in 4% paraformaldehyde for 10 minutes at 4°C and washed two times with PBS. Next sections were incubated in blocking buffer (PBS Tween 0.1% and 1% goat serum) for 1 hour at room temperature and subsequently incubated with primary antibody diluted in blocking buffer (1:20) overnight at 4°C. Primary antibodies used were: polyclonal rabbit anti-human Ki-67 (Ab15580 Abcam, Germany, Antibody Registry identifier AB_443209), polyclonal rabbit anti-human CD31 (Ab28364 Abcam, Germany, Antibody Registry identifier AB_726362) and monoclonal mouse anti-human nuclei (HuNu) (MAB1281 Millipore, Germany, Antibody Registry identifier AB_94090). The following day, sections were washed three times for 5 minutes with PBS and secondary antibody was added to the sections and incubated for 1 hour at room temperature. The sections were then washed in PBS 0.1% Tween three times for 5 minutes and then mounted with Vectashield (Biozol, Germany) and imaged using a fluorescence microscope (Zeiss Cell Observer-Z1, Germany).
7
Drosophila Indirect Flight Muscle Morphology
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Drosophila strains were grown under standard conditions at 27ºC to enhance GAL4 activity. All RNAi knock-downs were induced with Mef2-GAL4, a GAL4 line specifically expressed during development of all muscle types of the fly (58) . UAS RNAi lines used were from the Vienna (59) and Harvard collections (60) and ordered from VDRC or Bloomington stock centres. See Supplementary Table S6 for all genotypes used. The flight test was done as described in (58) .
Indirect flight muscle morphology was analysed in 3 to 5 days adult males or in 90 h APF pupae as previously published (61) . Briefly, 90 h APF pupae or adult flies were fixed with 4% paraformaldehyde in PBT (PBS, 0.5% triton X-100) and cut into half-thoraces using a sharp microtome blade. The half-thoraces were blocked with 3% normal goat serum in PBT for 30 min and F-actin in the flight and leg muscles was visualised with phalloidin (coupled to Alexa488 or rhodamine, Molecular Probes, 1:1000 in PBT for 2 h or overnight) and Sls was stained with anti-Kettin antibody MAC155 (Babraham Institute, 1:100 in PBT overnight). The stained half-thoraces were imbedded in Vectashield (Biozol) and imaged on an LSM780 or LSM880 confocal microscope. Images were processed using Fiji (62) .
Indirect flight muscle morphology was analysed in 3 to 5 days adult males or in 90 h APF pupae as previously published (61) . Briefly, 90 h APF pupae or adult flies were fixed with 4% paraformaldehyde in PBT (PBS, 0.5% triton X-100) and cut into half-thoraces using a sharp microtome blade. The half-thoraces were blocked with 3% normal goat serum in PBT for 30 min and F-actin in the flight and leg muscles was visualised with phalloidin (coupled to Alexa488 or rhodamine, Molecular Probes, 1:1000 in PBT for 2 h or overnight) and Sls was stained with anti-Kettin antibody MAC155 (Babraham Institute, 1:100 in PBT overnight). The stained half-thoraces were imbedded in Vectashield (Biozol) and imaged on an LSM780 or LSM880 confocal microscope. Images were processed using Fiji (62) .
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