Király K, Lammi M, Arokoski J, Lapveteläinen T, Tammi M, Helminen H, Kiviranta I
Department of Anatomy, University of Kuopio, Finland.
Histochem J. 1996 Feb;28(2):99-107. doi: 10.1007/BF02331414.
The ability of Safranin O, added to fixation and decalcification solutions, to prevent the escape of glycosaminoglycans (GAGs) from small cartilage tissue blocks during histological processing of cartilage has been studied. GAGs in the fixatives and decalcifying solutions used and those remaining in the 1 mm3 cubes of cartilage were assayed biochemically. The quantity of GAGs remaining in the cartilage cubes were determined from Safranin O-stained sections using videomicroscopy or microspectrophotometry. A quantity (10.6%) of GAGs were lost during a conventional 4% buffered formaldehyde fixation (48 h) and a subsequent decalcification in 10% EDTA (12 days) at 4 degrees C. Roughly one-quarter of the total GAG loss occurred during the 48 h fixation, and three-quarters during the 12 days of decalcification. Inclusion of 4% formaldehyde in the decalcification fluid decreased the loss of GAGs to 6.2%. The presence of 0.5% Safranin O in the fixative reduced this loss to 3.4%. When 0.5% Safranin O was included in the fixative and 4% formaldehyde in the decalcification solution, Safranin O staining of the histological sections increased on average by 13.5%. After fixation in the presence of 0.5% Safranin O, there was no difference in the staining intensities when decalcification was carried out in the presence of either Safranin O or formaldehyde, or both. It took 24 h for Safranin O to penetrate into the deep zone of articular cartilage, warranting a fixation period of at least this long. In conclusion, the addition of Safranin O to the fixative and either Safranin O or formaldehyde in the following decalcification fluid, markedly reduces the loss of GAGs from small articular cartilage explants during histological processing. However, for immunohistochemical studies, Safranin O cannot be included in the processing solutions, because it may interfere.
研究了在固定液和脱钙液中添加番红O，在软骨组织学处理过程中防止糖胺聚糖（GAGs）从小软骨组织块中逸出的能力。对所用固定液和脱钙液中的GAGs以及残留在1立方毫米软骨块中的GAGs进行了生化分析。使用视频显微镜或显微分光光度法，根据番红O染色切片确定残留在软骨块中的GAGs数量。在4℃下，采用传统的4%缓冲甲醛固定（48小时）并随后在10%乙二胺四乙酸（EDTA）中脱钙（12天）过程中，有10.6%的GAGs丢失。GAGs总损失的约四分之一发生在48小时的固定过程中，四分之三发生在12天的脱钙过程中。在脱钙液中加入4%甲醛可将GAGs损失降至6.2%。固定液中含有0.5%番红O可将这种损失降至3.4%。当固定液中含有0.5%番红O且脱钙液中含有4%甲醛时，组织学切片的番红O染色平均增加13.5%。在含有0.5%番红O的固定液中固定后，在含有番红O或甲醛或两者的情况下进行脱钙时，染色强度没有差异。番红O需要24小时才能渗透到关节软骨的深层区域，因此固定时间至少需要这么长。总之，在固定液中添加番红O以及在随后的脱钙液中添加番红O或甲醛，可显著减少组织学处理过程中小关节软骨外植体中GAGs的损失。然而，对于免疫组织化学研究，处理液中不能加入番红O，因为它可能会产生干扰。
