Schiff试剂又称雪夫试剂、希夫试剂等,是Schiff于1866年发现的一种有效的醛试剂,用于检测醛基(-CHO),Schiff试剂主要成分是副品红碱,为三氨基三苯甲烷的氯化物,其中的醌基是品红显色原因,在酸化的品红液中加入亚硫酸或者亚硫酸盐,品红被还原为品红-亚硫酸(或称白亚磺酸),即Schiff试剂。
Schiff试剂主要用于糖原、黏液物质等染色,常与过碘酸(也称高碘酸)配合使用,过碘酸是一种强氧化剂,它能氧化糖类及有关物质中的1,2-乙二醇基,使之变为二醛,醛与Schiff试剂能结合成一种品红化合物,产生紫红色,配制好的Schiff试剂接近于无色,一般变黄或粉红应弃用。该试剂仅用于科研领域,不适用于临床诊断或其他用途。
自备材料:
1、10%福尔马林固定液、蒸馏水、过碘酸溶液、苏木素染色液、酸性乙醇分化液
2、系列乙醇、二甲苯或环保浸蜡脱蜡透明液
操作步骤(仅供参考):
1、常规固定,常采用10%福尔马林固定液,常规脱水包埋。
2、石蜡切片二甲苯或脱蜡透明液脱蜡入蒸馏水;冰冻切片直接入蒸馏水。
3、自来水冲洗2~3min,再用蒸馏水浸洗2次。
4、切片入过碘酸溶液,室温放置5~8min,一般不宜超过10min。
5、自来水冲洗1次,再用蒸馏水浸洗2次。
6、切片入Schiff 试剂室温阴暗处浸染10~20min,自来水冲洗10min。
7、入苏木素染色液,染细胞核1~2min,酸性乙醇分化液分化2~5s。
8、自来水冲洗10~15min后,更换蒸馏水清洗使其返蓝。
9、逐级常规乙醇脱水,二甲苯或脱蜡透明液透明,中性树胶封固。
染色结果:
PAS反应阳性物质(糖原或多糖):红色或紫红色
阴性对照(可选)
1、取淀粉酶1g溶解于PBS (pH5.3)100ml,处理30~60min,与其他切片共同入过碘酸溶液,结果应为阴性。
2、(备选方案)取唾液片(过滤后用)处理30~60min,与其他切片共同入过碘酸溶液,结果应为阴性。
3、(备选方案)如果对照片采用其自身样本,对照片不经过碘酸溶液这一步,直接入Schiff 试剂,结果应为阴性。
注意事项
1、Schiff 试剂置于4°C密闭保存,使用时避免接触过多阳光和空气,在室温过低(小于15°C)情况下反应缓慢,可以置于适宜温水(小<30°C)中恢复后再使用。
2、理想的Schiff 试剂接近于无色,如果试剂颜色呈极轻微粉红色亦可考虑使用,如试剂颜色呈明显的红色应弃用。
3、颜色深浅很大程度上取决于样品在过碘酸溶液和Schiff 试剂中作用时间的长短。
4、为了您的安全和健康,请穿实验服并戴一次性手套操作。
5、试剂开封后请尽快使用,以防影响后续实验效果。
Schiff Reagent is an effective aldehyde reagent discovered by Schiff in 1866. It is used for the detection of aldehyde groups (-CHO). The main component of Schiff Reagent is parafuchsin, a chloride salt of triaminotriphenylmethane. The quinone group in parafuchsin is responsible for its color development. When sulfurous acid or sulfite is added to an acidified fuchsin solution, fuchsin is reduced to fuchsin-sulfurous acid (leucofuchsin sulfonic acid), which is Schiff’s reagent.
Schiff Reagent is mainly used for the staining of glycogen, mucous substances, and other structures. It is often used in combination with periodic acid. Periodic acid is a strong oxidizing agent that oxidizes the 1,2-glycol groups in carbohydrates and related compounds to form dialdehydes. The aldehydes react with Schiff Reagent to form a fuchsin-colored compound, producing a purplish red color. Freshly prepared Schiff Reagent is nearly colorless. It should be discarded if it turns yellow or pink. This reagent is for research use only. Not for clinical diagnosis or other purposes.
Materials Required:
1. 10% formalin fixative, distilled water, periodic acid solution, hematoxylin staining solution, acid ethanol differentiation solution
2. Graded ethanol series, xylene or ecofriendly dewaxing and clearing agent
Protocol (for reference only):
1. Routine fixation, typically with 10% formalin fixative, followed by routine dehydration and embedding.
2. Deparaffinize paraffin sections in xylene or dewaxing agent and transfer to distilled water; frozen sections go directly into distilled water.
3. Rinse in running tap water for 2–3 minutes, then immerse and wash twice in distilled water.
4. Place sections in periodic acid solution and incubate at room temperature for 5–8 minutes; generally not longer than 10 minutes.
5. Rinse once in tap water, then immerse and wash twice in distilled water.
6. Stain sections in Schiff’s reagent in the dark at room temperature for 10–20 minutes, then rinse in running tap water for 10 minutes.
7. Stain nuclei in hematoxylin staining solution for 1–2 minutes, differentiate in acid ethanol differentiation solution for 2–5 seconds.
8. Rinse in running tap water for 10–15 minutes, then wash in distilled water to blue the nuclei.
9. Dehydrate through graded ethanol series, clear in xylene or dewaxing agent, and mount with neutral balsam.
Staining Results:
PAS-positive substances (glycogen or polysaccharides): Red or purplish red
Negative Control (Optional):
1. Dissolve 1 g amylase in 100 ml PBS (pH 5.3), treat sections for 30–60 min, then incubate with other sections in periodic acid solution. The result should be negative.
2. (Alternative) Treat sections with filtered saliva for 30–60 min, then incubate with other sections in periodic acid solution. The result should be negative.
3. (Alternative) Use adjacent sections from the same sample. Incubate control sections directly in Schiff’s reagent without periodic acid oxidation. The result should be negative.
Precautions:
1. Store Schiff’s reagent at 4 °C in a tightly sealed container. Avoid prolonged exposure to light and air during use. The reaction is slow at room temperature below 15 °C; warm the reagent gently in water (< 30 °C) before use.
2. Fresh Schiff’s reagent is nearly colorless. Slightly pink reagent may still be used, but obviously red reagent should be discarded.
3. Staining intensity largely depends on the incubation time in periodic acid solution and Schiff’s reagent.
4. For your safety and health, wear laboratory coat and disposable gloves during operation.
5. Use the reagent promptly after opening to avoid affecting experimental results.