Semiquantitative AMPK Immunohistochemistry

For AMP-activated protein
kinase semiquantitative assessment, the
slides were incubated overnight at 4 °C with primary AMPK antibodies
in the concentrated form consisting of rabbit IgG in phosphate buffer
saline (free from Mg²⁺ and Ca²⁺), pH 7.4, 150
mM NaCl, 0.02% sodium azide, and 50% glycerol. Detection was carried
out using a Vector ABC kit (Vector Laboratories, CA) and DAB reagent
(Dako Comp, Japan). All slides were examined as described in Section 2.3.4.3.
The proportions of positive cells were also calculated, and ranges
were assigned from 10 to 100%. The intensity of staining was given
scores of 0 (negative), 1 (very weak), 2 (weak), 3 (moderate), and
4 (intense). Lastly, the immunoreactivity score was calculated as
a percentage of positive cells multiplied by the intensity of staining.^{58 (link)}