Originals
The newly developed double staining of growth-line and osteoid matrix in paraffin-embedded decalcified hard tissues
1985 Volume 10 Issue 2 Pages 78-84
Details
1985 Volume 10 Issue 2 Pages 78-84
An improved double staining method was discussed for labeling of growth lines in hard tissues as well as identification of osteoid matrix simultaneously. For the experimental study, following procedures were made.
Male Wistar rats were injected subcutaneously with 1.0%lead-disodium-EDTA (30mg/kg), followed by a second injection five days later. Five days after the second injection, the animals were sacrificed by ether anesthesia. Their upper and lower jaws and femur were dissected and fixed in 10%neutral buffered formalin immediately. The specimens were cut in 3-5mm thickness to observe the staining quality of growth lines and osteoid matorix as well. After these procedures, two experimental studies were made.
The first study was made for labeling of growth lines in hard tissues. Specimens were demineralized in a 10% EDTA solution containing 5% sodium sulfide at 4°C for 2-3 weeks, After demineralization, they were submitted into block staining in 0.1% gold chloride solution for 60 minutes, and rinsed in 5% sodium thiosulfate solution for 20 minutes. After that, they were dehydrated in ascending ethanol, and embedded in paraffin. Then, 7μm thin sections were made. After deparaffinization, sections were stained with 0.1% gold chloride solution for 1-2 hours, and rinsed in 5% sodium thiosulfate solution for 20 minutes.
The second study was made for labeling of growth lines in hard tissues and osteoid matrix. Specimens were dehydrated in ascending ethanol. And then, they were immersed in freshly prepared 0.5% cyanuric chloride in anhydrous methanol containing 1.0% N-methyl morpholine for approximately two days at room temparature. Remaining procedures for the staining were the same of that first study.
Growth lines in hard tissues were clearly observed with no disadvantageous effects caused by the dehydrating in the first study. In the second study, labeling lines in hard tissues and osteoid matrix were observed in the paraffin-embedded specimens simultaneously.
