The Intra‐cellular Location of Disulfide‐bond‐rich Particles

Four micron cryostat sections of three‐day‐old rat skin were fixed in 98% ethanol at −20°C for 4 minutes. These sections were preincubated in 10mM phosphate buffered saline, pH 7.4, containing 10mM 2‐mercaptoethanol at 37°C for 30 minutes and washed thoroughly in the same buffer. After all the free sulfhydryl groups were blocked with N‐ethylmaleimide and disulfide bonds were again reduced with 2‐mercaptoethanol, the section was reacted with 7‐(N‐dimethylamino‐4‐methyl‐3‐coumarinyl) maleimide. Many minute fluorescent particles were seen in the stratum granulosum of the epidermis, especially of the acrotrichium. Cryostat sections containing an acrotrichium were fixed again in 2.5% glutaraldehyde and embedded in Epon. Thin sections were examined under the electron microscope. The location of the disulfide bond‐rich particles around an acrotrichium was examined and compared with the location of these fluorescent particles. These disulfide‐bond‐rich particles were found to be low electron dense particles 0.3 to 0.7 μm in diameter located in or around the keratohyalin granules.