Cowden R R, Rasch E M, Curtis S K
Histochemistry. 1976 Aug 12;48(2):81-92. doi: 10.1007/BF00494546.
Appropriately fixed preparations stained by a modification of the Guard (1959) reaction for "sex chromatin" display selective staining of interphase chromatin and mitotic or meiotic chromosomes. This is a regressive staining method which seems to depend on the selective displacement of an acidic dye from less basic structures, and retention of the dye at more basic sites. The results obtained with the reaction can be controlled by the length of time that the preparations are "differentiated" in solutions containing phosphomolybdic and phosphotungstic acids (polyacids). After three- or four-hour exposures to polyacid solutions, all chromatin is stained. However, with longer differentiation, "condensed" chromatin can be stained preferentially. Of a number of fixatives investigated, only 10% formalin, ethanol-acetic acid (3:1), and Bouin's solution proved useful. Others resulted in diminished specificity or a total loss of selectivity. The most intense staining was obtained after formalin fixation. Less intense dyebinding was observed after fixation in 3:1 - probably due to extraction of some histone fractions-and the least amount of dye was bound in Bouin's-fixed chromatin - probably due to blockage of arginine residues by picric acid. The reaction was not affected by enzymatic removal of nucleic acids or the extraction of lipids. It was diminished by treatment with trypsin or weak acetylation, and it was completely prevented by strong acetylation, deamination, or extraction of basic proteins with HCl. The results presented suggest that the modified Guard (1959) procedure selectively demonstrates basic nucleoproteins. Further, by the use of regressive differentiation in polyacid solutions, the retention of dye in more condensed chromatin can be favored.
用改良的瓜德（1959年）“性染色质”反应染色的适当固定制剂，能显示间期染色质以及有丝分裂或减数分裂染色体的选择性染色。这是一种退行性染色方法，似乎取决于酸性染料从碱性较弱结构中的选择性置换，以及染料在碱性较强位点的保留。该反应得到的结果可通过制剂在含磷钼酸和磷钨酸（多酸）溶液中“分化”的时间长短来控制。在多酸溶液中暴露三到四个小时后，所有染色质都被染色。然而，分化时间更长时，“浓缩”染色质可被优先染色。在所研究的多种固定剂中，只有10%福尔马林、乙醇 - 乙酸（3:1）和波因氏液被证明是有用的。其他固定剂导致特异性降低或选择性完全丧失。福尔马林固定后染色最强。3:1固定后观察到染料结合较弱——可能是由于一些组蛋白组分被提取——而波因氏固定的染色质中结合的染料量最少——可能是由于苦味酸对精氨酸残基的封闭。该反应不受核酸酶解或脂质提取的影响。用胰蛋白酶处理或轻度乙酰化会使其减弱，而强烈乙酰化、脱氨基或用盐酸提取碱性蛋白则会完全阻止该反应。所呈现的结果表明，改良的瓜德（1959年）方法能选择性地显示碱性核蛋白。此外，通过在多酸溶液中进行退行性分化，可使染料在更浓缩的染色质中保留得更好。
