Serial sections from previously DMM-operated C57BL/6 mice were stained with safranin O according to standard protocols [25 (link)]. Cartilage immunostaining was performed on 5 μm thick sections, which were deparaffinised, hydrated, antigen retrieved and blocked as previously described [22 (link)]. Sections were incubated overnight at 4 °C with Cy5.5-1-11E-scFv or control Cy5.5-C7-scFv (10 μg/ml) in DAKO antibody diluent solution (Dako, Cambridge, UK). The pericellular matrix was stained using the rat anti-heparan sulphate proteoglycan antibody (Millipore, Watford, UK) followed by the Alexa-Fluor-488 labelled anti-rat IgG (Life Technologies, Paisley, UK). Slides were viewed under the LSM 510 Meta (Zeiss, Cambridge, UK) using the 488 nm excitation laser to visualize the Alexa-488 label and the 633 nm excitation laser to visualize the Cy5.5-label.
Dako antibody diluent solution
Manufactured by Agilent Technologies
Sourced in United Kingdom, United States
DAKO antibody diluent solution is a laboratory reagent used to dilute antibodies prior to their application in various immunohistochemical and immunocytochemical procedures. The solution is designed to maintain the stability and performance of the diluted antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (2 mentions)
Lsm 510 confocal system, by Zeiss (2 mentions)
Tissue tek optimal cutting temperature compound, by Sakura Finetek (2 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Oregon green 488, by Thermo Fisher Scientific (1 mentions)
Fluoro gel mounting medium, by Electron Microscopy Sciences (1 mentions)
16 well chamber slide, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Agilent Technologies (1 mentions)
Tcs nt sp, by Leica (1 mentions)
Triton x 100, by Merck Group (1 mentions)
5 protocols using dako antibody diluent solution
1
Cartilage Immunostaining in C57BL/6 Mice
2
Immunofluorescence Staining Protocol
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5 × 10 [3 (link)] cells were plated into 16 well chamber slides (Bio-Tek, Nunc, Winooski, VT), fixed with methanol/ethanol 1:1 vol for 5 min, and blocked with 5% goat serum (Thermo Fisher; Minneapolis, MN). Subsequently, slides were incubated with primary antibody in Dako antibody diluent solution (Dako, Camarillo, CA) for 1 h at room temp. Slides were washed with 1X TBS-T (Dako, Camarillo, CA), then incubated with secondary antibody (anti-rabbit Oregon green 488 or anti-mouse Alexa red 594 from Invitrogen, Carlsbad, CA) in the dark for 1 h at room temp. Slides were washed with 1X TBS-T and double deionized water and subsequently counterstained with DAPI (1 μg/ml, Santa Cruz Biotechnology, Santa Cruz, CA). Slides were mounted using Fluorogel mounting medium (Electron Microscopy Sciences, Hatfield, PA). Fluorescence microscopy was performed using Zeiss microscope and AxiovisionRel 4.8 software.
3
Confocal Analysis of Rsad2 Protein
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Confocal analysis was conducted as previously described24 (link). After fixation for 10 min in 4% paraformaldehyde in PBS, cells were washed in 1% Tween20/PBS and permeabilized with 0.1% Triton-X 100 (Sigma-Aldrich) in PBS. Cells were then incubated in 5% NGS in room temperature for blocking. After washing with 1% Tween20 in PBS, cells were incubated for 24 h at 4 °C with an anti-Rsad2 antibody (Abcam). After further washing in 1% Tween20 in PBS, cells were stained for 2 h at room temperature with a secondary antibody (Alexa fluor 488 or 555-conjugated anti-mouse Ab (DAKO, Santa Clara, CA, USA)) in DAKO antibody diluent solution (DAKO). Cells were then resuspended in DAPI (DAKO). Confocal microscopy was performed using a Leica TCS-NT SP equipped with argon, krypton, and helium/neon lasers, and a spectrophotometer was used to separate the detection channels of Alexa fluor488 (450–500 nm) and Alexa fluor555 (580–660 nm).
4
Immunostaining Lung Tissue and Endothelial Cells
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Lung tissues were fixed with 4% paraformaldehyde and dehydrated with a 15% sucrose solution followed by 30% sucrose solution until the lung tissues were settled at the bottom. Tissues were then embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA), and 5-μm thin sections were cut. The sections were fixed in ice-cold acetone for 10 minutes, blocked with Dako antibody diluent solution (Agilent, Santa Clara, CA), and stained overnight at 4 °C with rat anti-mouse CD31 antibody (5 µg/mL), rabbit polyclonal anti-murine TF antibodies (5 µg/mL), and murine anti-human α-smooth muscle actin (α-SMA; 5 µg/mL, cross-reacts with murine α-SMA), followed by Alexa-488-, Alexa-594-, and Alexa-647-conjugated secondary antibodies. The nuclei were stained with DAPI (5 µg/mL). To immunostain cultured endothelial cells, the cells were fixed in 2% paraformaldehyde and stained with antibodies against goat anti-human VE-cadherin (5 µg/mL) and EPCR (endothelial cell protein C receptor) mAb (JRK1500, 5 µg/mL); the nuclei were stained with DAPI. Confocal images were obtained using an LSM 510 confocal system (Carl Zeiss). Immunostained tissue sections or cells were viewed using a Plan-APOCHROMAT 63.3/1.4 NA oil objective lens.
5
Multicolor Immunofluorescence Staining of Lung Tissues
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