Neutrophils (concentration of 1 × 106 cells suspended in 100 μL RPMI medium with 10% FBS) were seeded onto glass coverslips treated with 0.001% poly-l-lysine (Matsunami glass, Tokyo, Japan) and placed in a 35 mm dish (Iwaki, Shizuoka, Japan). Cells were incubated for 1 h at 37 °C in 5% CO2. Neutrophils were incubated with PMA for 30 min to induce NETs formation (or with PBS for control), and then, 107 CFU octadecyl rhodamine B chloride (Sigma-Aldrich Corp.) labeled live or heat-killed M. bovis (or with PBS for control) were added and incubated for 30 min at 37 °C under a 5% CO2. Neutrophils were washed with PBS and stained with 4,6-diamidino-2-phenylindole, dilactate (DAPI) for 15 min (Dojindo, Tokyo, Japan). Coverslips were washed with PBS, coated with Fluoromount (Diagnostic Biosystems, Pleasanton, CA, USA), and viewed using a fluorescence microscope (Nikon, Tokyo, Japan). Three bovine neutrophil studies were performed individually.
4 6 diamidino 2 phenylindole dilactate dapi
Manufactured by Dojindo Laboratories
Sourced in Japan
4,6-diamidino-2-phenylindole, dilactate (DAPI) is a fluorescent dye that binds to DNA. It is commonly used in biological research applications to stain and visualize cell nuclei.
Automatically generated - may contain errors
2 protocols using 4 6 diamidino 2 phenylindole dilactate dapi
1
Neutrophil-M. bovis Interaction Assay
2
Immunofluorescence Analysis of S. uberis Mastitis
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Milk clots in mastitic milk were obtained from three udders of three Holstein cows
infected with S. uberis NH386. Milk clots were provisionally embedded in
2% (w/v) low melting point agarose (24–28°C gelling temperature) (Agarose LM: Naclai
Tesque, Kyoto, Japan), fixed with buffered formalin, embedded in paraffin, and sectioned
at 4 µm. Tissue sections were stained using hematoxylin and eosin. Immunofluorescence
microscopy was performed as described below. The dried sections were deparaffinized in
xylene. Antigen retrieval was performed in citrate buffer (0.37 g/mL of citric acid and
2.4 g/mL of trisodium citrate dihydrate) by microwave heating for 9 min. The sections were
blocked for 1 hr at room temperature and incubated using the rabbit antiserum against
S. uberis for 1 hr at room temperature. Sections that were washed twice
using PBS were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG heavy and
light chains (Abcam, Cambridge, MA, USA) at room temperature for 1 hr. The sections were
washed twice using PBS and incubated with 4′, 6-diamidino-2-phenylindole dilactate (DAPI;
Dojindo, Tokyo, Japan) at room temperature for 30 min. Sections were examined using a
ZEISS Axio Observer microscope (Carl Zeiss, Oberkochen, Germany).
infected with S. uberis NH386. Milk clots were provisionally embedded in
2% (w/v) low melting point agarose (24–28°C gelling temperature) (Agarose LM: Naclai
Tesque, Kyoto, Japan), fixed with buffered formalin, embedded in paraffin, and sectioned
at 4 µm. Tissue sections were stained using hematoxylin and eosin. Immunofluorescence
microscopy was performed as described below. The dried sections were deparaffinized in
xylene. Antigen retrieval was performed in citrate buffer (0.37 g/mL of citric acid and
2.4 g/mL of trisodium citrate dihydrate) by microwave heating for 9 min. The sections were
blocked for 1 hr at room temperature and incubated using the rabbit antiserum against
S. uberis for 1 hr at room temperature. Sections that were washed twice
using PBS were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG heavy and
light chains (Abcam, Cambridge, MA, USA) at room temperature for 1 hr. The sections were
washed twice using PBS and incubated with 4′, 6-diamidino-2-phenylindole dilactate (DAPI;
Dojindo, Tokyo, Japan) at room temperature for 30 min. Sections were examined using a
ZEISS Axio Observer microscope (Carl Zeiss, Oberkochen, Germany).
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