半薄切片是可供光学显微镜观察研究的塑料切片技术制片，方法与超薄切片相同 (切片厚度 0.1μm 以下，通常 60-80nm)，但半薄切片厚度约在 0.5-1.5 μm，介于石蜡切片与超薄切片之间；效果比石蜡切片要好，因为较薄，所以能较好的展示细胞的结构，减少人为假象，成像效果比石蜡切片更加清晰。多用于神经组织切片观察。
半薄切片染色液 (甲苯胺蓝法) 由甲苯胺蓝、硼酸盐、去离子水等组成。由于硼酸盐缓冲液呈强碱性，更利于组织细胞的着色。横切半薄切片甲苯胺蓝染色，可在光学显微镜下观察神经髓鞘的病变情况及髓鞘的厚度；也可用于植物叶片、花药等，观察细胞排列情况等。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、蒸馏水或去离子水、系列乙醇、1% 锇酸固定液、0.1M PB 磷酸缓冲液 (pH7.4)
2、丙酮、812 包埋剂、二甲苯或环保脱蜡透明液、中性树胶
3、包埋板、烤箱、半薄切片机、载玻片、显微镜
操作步骤 (仅供参考):
1、组织用 0.1M PB 磷酸缓冲液 (pH7.4) 配制的 1% 锇酸避光室温固定 7h。0.1M PB 磷酸缓冲液 (pH7.4) 漂洗 3 次，每次 15min。
2、室温脱水：组织依次入 30%-50%-70%-80%-95%-100%-100% 酒精脱水，每次 1h。
3、无水乙醇：丙酮 (3:1) 30min，无水乙醇：丙酮 (1:1) 30 min，无水乙醇：丙酮 (1:3) 30min，丙酮 1h。
4、渗透：丙酮：812 包埋剂 (3:1) 37℃ 2-4h，丙酮：812 包埋剂 (1:1) 37℃渗透过夜，丙酮：812 包埋剂 (1:3) 37℃ 2-4h，纯 812 包埋剂 37℃ 5-8h。
5、包埋：将纯 812 包埋剂倒入包埋板，将样品插入包埋板后 37℃ 烤箱过夜包埋。
6、聚合：包埋板放于 60℃ 烤箱聚合 48h，取出树脂块备用。
7、切片：半薄切片机切片，厚度 1.5μm，组织片贴附于载玻片上。
8、染色：将甲苯胺蓝染液放在 60℃ 烤箱加热 1h，将片子放入染液中染 2min。
9、水洗，95% 酒精分化，在光镜下控制颜色，烤干，中性树胶封片后全景扫描。
注意事项:
1、标本包埋面不能过大，否则容易造成皱褶。
2、染色时宜加热条件下进行，利于染料穿透树脂。
3、半薄切片可采用相差法检查，也可染色后在光学显微镜下观察。
4、半薄切片需要用玻璃刀，不要用钻石刀。
5、四氧化锇 (锇酸) 蒸汽也有固定组织的作用，因此在配制锇酸固定液时需佩戴好手套和护目镜做好防护。经醛固定的标本在进入锇酸之前须用缓冲液清洗干净，防止两种固定液之间发生相互作用。
6、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Semi-thin sectioning is a plastic sectioning technique used to prepare specimens for observation and study under a light microscope. The procedure is the same as that for ultrathin sectioning (section thickness below 0.1 μm, usually 60–80 nm), but semi-thin sections are approximately 0.5–1.5 μm thick, intermediate between paraffin sections and ultrathin sections. The results are better than those of paraffin sections because the sections are thinner, allowing better demonstration of cellular structures and reducing artifacts; the imaging quality is clearer than that of paraffin sections. It is often used for the observation of nerve tissue sections.
Semithin section staining solution (Toluidine blue method) consists of toluidine blue, borate, deionized water, etc. Because borate buffer is strongly alkaline, it is more conducive to staining tissue cells. Transverse semi-thin sections stained with toluidine blue can be used to observe lesions of the nerve myelin sheath and myelin thickness under a light microscope; they can also be used for plant leaves, anthers, etc., to observe cell arrangement, etc. This reagent is for research use only and is not suitable for clinical diagnosis or other purposes.
Operating Procedures (For reference only):
1. Distilled water or deionized water, graded ethanol, 1% osmic acid fixative, 0.1 M PB phosphate buffer (pH 7.4)
2. Acetone, 812 embedding medium, xylene or environmentally friendly dewaxing clearing solution, neutral balsam
3. Embedding plates, oven, semi-thin microtome, glass slides, microscope
Procedure (for reference only):
1. Fix tissue in 1% osmic acid prepared in 0.1 M PB phosphate buffer (pH 7.4) at room temperature in the dark for 7 h. Rinse with 0.1 M PB phosphate buffer (pH 7.4) 3 times, 15 min each.
2. Dehydrate at room temperature: sequentially transfer tissue into 30%, 50%, 70%, 80%, 95%, 100%, and 100% ethanol for dehydration, 1 h each.
3. Absolute ethanol:acetone (3:1) for 30 min, absolute ethanol:acetone (1:1) for 30 min, absolute ethanol:acetone (1:3) for 30 min, acetone for 1 h.
4. Infiltration: acetone:812 embedding medium (3:1) at 37°C for 2–4 h; acetone:812 embedding medium (1:1) at 37°C overnight; acetone:812 embedding medium (1:3) at 37°C for 2–4 h; pure 812 embedding medium at 37°C for 5–8 h.
5. Embedding: Pour pure 812 embedding medium into embedding plates, insert samples into the embedding plates, and embed overnight in a 37°C oven.
6. Polymerization: Place the embedding plates in a 60°C oven for polymerization for 48 h, then remove the resin blocks for later use.
7. Sectioning: Section with a semi-thin microtome at a thickness of 1.5 μm, and attach the tissue sections to glass slides.
8. Staining: Heat the toluidine blue staining solution in a 60°C oven for 1 h, then place the slides in the staining solution for 2 min.
9. Wash with water, differentiate in 95% ethanol, control the color under a light microscope, dry in an oven, mount with neutral balsam, and perform whole-slide scanning.
Precautions：
1. The embedding surface of the specimen should not be too large; otherwise, wrinkles are easily formed.
2. Staining should preferably be carried out under heating to facilitate penetration of the dye into the resin.
3. Semi-thin sections can be examined by phase contrast, or observed under a light microscope after staining.
4. Glass knives should be used for semi-thin sectioning; do not use diamond knives.
5. Osmium tetroxide (osmic acid) vapor also has a tissue-fixing effect. Therefore, when preparing osmic acid fixative, wear gloves and goggles for protection. Specimens fixed with aldehyde must be thoroughly washed with buffer before entering osmic acid to prevent interaction between the two fixatives.
6. For your safety and health, please wear a lab coat and disposable gloves during operation.
危险品化学品经营许可证（带存储）