Paraffin-embedded liver sections were cleared from paraffin using xylene and ethanol in ascending line and distilled water for all staining procedures. Hematoxylin and eosin (H&E) staining was done by 15 minutes of incubation in hematoxylin, rinsing for 10 minutes using warm tap water, and 5 minutes of staining with eosin. Sirius Red staining was performed using 100 mg Sirius Red (Polysciences Inc., Warrington, USA) in 100 mL saturated picric acid for staining, with the pH adjusted to 2 using 2 N NaOH. To differentiate, sections were incubated for two minutes in 0.01 N HCl and rinsed with tap water. Morphometric quantification of collagen fibres was done using Image J. Staining for α smooth muscle actin (αSMA) was done using the antibody M0851 and the Animal Research Kit, according to the instructions of the manufacturer (all Dako, Hamburg, Germany). All sections were dehydrated using a descending order of ethanol and xylene.
Animal research kit
Manufactured by Agilent Technologies
Sourced in Germany
The Animal Research Kit is a comprehensive set of laboratory equipment designed for various animal research applications. It includes essential tools and instruments necessary for procedures commonly performed in animal studies. The kit provides a standardized and organized solution to support research activities involving small laboratory animals.
Automatically generated - may contain errors
Lab products found in correlation
M0851, by Agilent Technologies (1 mentions)
Sirius red, by Polysciences (1 mentions)
3 3 diaminobenzidine substrate, by Agilent Technologies (1 mentions)
Blocking reagent, by Agilent Technologies (1 mentions)
M7240, by Agilent Technologies (1 mentions)
Bromodeoxyuridine (brdu), by Thermo Fisher Scientific (1 mentions)
Active caspase 3, by Cell Signaling Technology (1 mentions)
Api4000 q trap hybrid triple quadrupole linear ion trap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Agilent 1100 series liquid chromatography, by Agilent Technologies (1 mentions)
Prism 6, by GraphPad (1 mentions)
6 protocols using animal research kit
1
Histological Analysis of Liver Fibrosis
2
Immunohistochemistry and Immunofluorescence Protocols
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For immunohistochemistry frozen sections were fixed with 4% formalin at RT, blocked with peroxidase-block, washed in phosphate buffered saline (PBS) and blocked with a Streptavidin-Biotin block (both from Vector Laboratories), according to manufacturer's instructions. Then sections were blocked with 20% goat serum for 30 min at RT, incubated with the biotinylated anti-EpCAM antibody (using the Animal Research Kit (Dako), 1:200, 2 h, RT (Enzo Life Sciences), and horse radish peroxidase-labeled streptavidin (1:800, 20 min, RT).
For immunofluorescence FFPE samples were washed in PBS and permeabilized in 0.1% Triton-X, incubated with Cy3-labeled human anti-nuclei antibody (Merck Millipore, dilution 1:300) 1 h at 37 °C, washed in PBS and mounted in glycerol and PBS (1:1) containing 5 µg/mL 4′,6-Diamidin-2-phenylindole (DAPI) (Roth). Staining was visualized with 3,3′-Diaminobenzidine substrate (Dako) and counterstained with hematoxylin, then mounted with VectaMount Aqueous Mounting Medium.
For immunofluorescence FFPE samples were washed in PBS and permeabilized in 0.1% Triton-X, incubated with Cy3-labeled human anti-nuclei antibody (Merck Millipore, dilution 1:300) 1 h at 37 °C, washed in PBS and mounted in glycerol and PBS (1:1) containing 5 µg/mL 4′,6-Diamidin-2-phenylindole (DAPI) (Roth). Staining was visualized with 3,3′-Diaminobenzidine substrate (Dako) and counterstained with hematoxylin, then mounted with VectaMount Aqueous Mounting Medium.
3
Histological Analysis of Pancreata
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The removed pancreata were fixed in 4% buffered formaldehyde (Agar, Stansted, UK) for 24 h, embedded, cut into 4 µm thick sections and stained with hematoxylin and eosin (H&E). For IHC and IFM, sections were subjected to antigen retrieval by boiling in EDTA buffer. Blockings of endogenous peroxidases and unspecific antigen interactions were performed with 3% H2O2 and 2% BSA, respectively. Afterwards, sections were incubated with primary antibodies for 1 h at room temperature. Fluorophore-conjugated antibodies were used for IFM. Primary antibodies used in IHC were visualized by biotinylated secondary antibodies and diaminobenzidine (DAB) detection using the animal research kit (Dako).
4
Quantifying Tumor Cell Proliferation
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To determine the fraction of proliferating tumor cells, immunohistochemical staining was done with an antibody against human Ki67 (M7240, DAKO). 10 µm-thick tumor cryosections were fixed with a 4% PFA solution. Further treatment of the slides was performed according to the protocol already described for CD31 staining. The anti-human Ki67 antibody was applied to the sections at a concentration of 0.0016 mg/mL. The primary antibody was pretreated with a biotinylation reagent (Animal Research Kit, DAKO) for 20 min. In addition, the primary antibody was mixed with a blocking reagent (DAKO) for 20 min before administration to the cryosections. After incubation overnight at 4 °C, the avidin-biotin complex was applied to the tumor tissue sections for 30 min, which was later developed with AEC. The applied staining reaction was stopped after complete red staining of the proliferating Ki67 containing tumor cells using distilled water. Counterstaining with hematoxylin was used to improve the identification of the non-proliferating tumor cells. Counting of Ki67-labeled cells in 3 to 4 fields of view imaged at 200× magnification was performed using the ImageJ v145.
5
BrdU Labeling in NHERF1 Mice
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NHERF1 wild-type and mutant mice were injected intraperitoneally with 0.1 ml/10 g body weight of BrdU (Invitrogen), and sacrificed 2 hours later. Organs were harvested, fixed in 10% formalin overnight, and processed for IHC with the Animal Research Kit (Dako, Carpinteria, CA).
6
Sphingolipid Profiling in Lung Tissues
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