Dako envison kit hrp

Paraffin-embedded serial sections of liver tissues were stained immunohistochemically with anti-M-CSF (Santa Cruz Biotechnology, Santa Cruz, CA), anti-CD68 (DAKO, Kyoto, Japan), anti-CD163 (Abcam, Cambridge, United Kingdom), or anti-CD31 (Abcam) antibodies. Briefly, after deparaffinization and rehydration, the antigen retrieval procedure was applied by heating the slides in 0.1 mol/L citrate buffer (pH 6.0) for 10 min. The sections were then incubated first with 0.3% H₂O₂ in distilled water for 5 min to block endogenous peroxidase and then incubated with one of the following antibodies: monoclonal mouse antihuman CD68 (diluted to 1:200), CD163 (diluted to 1:200), CD31 (diluted to 1:200) , and M-CSF (diluted to 1:100) at 4 °C overnight. Biotinylated secondary antibody conjugated with avidin-biotin horseradish peroxidase (Dako Envison kit/HRP; Dako, Kyoto, Japan) and 3’,3’-diaminobenzidine tetrahydrochloride were used for standard avidin biotinylated peroxidase detection. Quantitation of cells that stained positive for CD68, CD163, CD31 and M-CSF was performed using image analysis software (Scion Image; Scion Corp., Frederick, MD) by evaluating five different fields (magnification: × 400) for areas positively stained and expressed as a percentage of the total area.