Whole lungs with trachea were removed, immersion fixed in 4% paraformaldehyde pH 7.0 (Carl Roth, Karlsruhe, Germany) for 48 hours and embedded in paraffin (Thermo Scientific, Waltham, MA). 4 µm thick whole-lung horizontal sections were stained with hematoxylin & eosin (Carl Roth, Karlsruhe, Germany) and scored in a blinded fashion by a veterinary pathologist for the following parameters: perivascular edema, pleuritis, necrosis, area of infection. For immunohistochemical detection of S. pneumoniae in paraffin embedded sections, antigen retrieval was performed with microwave heating (600 W) for 12 minutes in 750 ml 10 mM citric acid pH 6.0 (Merck, Darmstadt, Germany). Sections were incubated with a purified rabbit polyclonal antibody which recognizes epitopes in the capsule, the cell wall and the cytosol of S. pneumoniae (1:2000, kindly provided by Sven Hammerschmidt, Interfaculty Institute for Genetics and Functional Genome Research, University of Greifswald, Germany) at 4°C overnight and subsequently incubated with an alkaline phosphatase conjugated goat anti-rabbit secondary antibody (1:500, Vector, Burlingame, CA) for 30 minutes at room temperature as described in (18 (link)).
Alkaline phosphatase conjugated goat anti rabbit secondary antibody
Manufactured by Vector Laboratories
Sourced in Germany, United States
Alkaline phosphatase conjugated goat anti-rabbit secondary antibody is a laboratory reagent used for the detection and visualization of target proteins in immunoassays. The antibody is produced in goats and is specific to rabbit primary antibodies, with alkaline phosphatase enzyme conjugated to facilitate colorimetric or chemiluminescent signal generation.
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2 protocols using alkaline phosphatase conjugated goat anti rabbit secondary antibody
1
Immunohistochemical Analysis of Pneumococcus
2
Flagella Protein Separation and Analysis
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Flagella, KCl extract, KI extract, and residual axonemes were loaded on to a 4-15% SDS-polyacrylamide gel and separated by electrophoresis. The resulting protein bands were then transferred onto a polyvinylidene fluoride (PVDF) membrane using a Bio-Rad mini-gel transfer apparatus, followed by incubation with a blocking buffer (3% skim milk in phosphate-buffered saline with 0.05% Tween-20) for an hour at 20-25°C. The PVDF membrane was incubated for 45 min with the anti-FAP85 antibody. After washing three times with the blocking buffer, the membrane was incubated for 45 min with an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA) for Sarkosyl experiments, and IgG, horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (GE Healthcare, Tokyo, Japan) for other experiments. The membrane was then washed three times with blocking buffer and stained with the BCIP/NBT phosphatase substrate system (Sera Care Life Sciences, Inc., Milford, MA, USA) or the ECL substrate (Clarity Wester ECL Substrate, Bio-Rad).
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