Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (9): 2554-2564.doi: 10.16420/j.issn.0513-353x.2024-0849
• New Technology and New Method • Previous Articles Next Articles
WU Chunhao, YAN Xingkai, LU Mingyan, ZHAO Ying, ZHANG Maojun, LI Siyu, WANG Qiang*()
Received:
2025-03-17
Revised:
2025-07-07
Online:
2025-09-25
Published:
2025-09-24
Contact:
WANG Qiang
WU Chunhao, YAN Xingkai, LU Mingyan, ZHAO Ying, ZHANG Maojun, LI Siyu, WANG Qiang. Optimization of Paraffin Section Preparation Method for Pear Tissues[J]. Acta Horticulturae Sinica, 2025, 52(9): 2554-2564.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2024-0849
步骤 Step | 试剂梯度 Reagent gradient | 叶片 Leaf | 新梢 Shoot | 花芽 Flower bud | |||
---|---|---|---|---|---|---|---|
CPSM | MWBM | CPSM | MWBM | CPSM | MWBM | ||
脱水 Dehydration | 70%乙醇 70% Ethanol | 60 | 1 | 120 | 6 | 120 | 6 |
85%乙醇 85% Ethanol | 60 | 1 | 120 | 6 | 120 | 6 | |
95%乙醇 95% Ethanol | 60 | 1 | 120 | 6 | 120 | 6 | |
100%乙醇 100% Ethanol | 60 | 1 | 120 | 6 | 120 | 6 | |
100%乙醇 100% Ethanol | 60 | 1 | 120 | 6 | 120 | 6 | |
透明 Clearing | 1/2乙醇 + 1/2二甲苯 1/2 Ethanol + 1/2 Xylene | 60 | 1 | 120 | 6 | 120 | 6 |
二甲苯 Xylene | 60 | 1 | 120 | 6 | 120 | 6 | |
二甲苯 Xylene | 60 | 1 | 120 | 6 | 120 | 6 | |
浸蜡 Dipping paraffin | 1/2二甲苯+石蜡 1/2 Xylene + Paraffin | 90 | 10 | 120 | 15 | 120 | 15 |
石蜡 Paraffin | 90 | 10 | 120 | 15 | 120 | 15 | |
石蜡 Paraffin | 90 | 10 | 120 | 15 | 120 | 15 | |
石蜡 Paraffin | — | 10 | — | 15 | — | 15 | |
总时长 Total time | 750 | 48 | 1 320 | 108 | 1 320 | 108 |
Table 1 Comparison of treatment time between conventional paraffin sectioning(CPSM)and microwave and water bath(MWBM)in different pear tissues min
步骤 Step | 试剂梯度 Reagent gradient | 叶片 Leaf | 新梢 Shoot | 花芽 Flower bud | |||
---|---|---|---|---|---|---|---|
CPSM | MWBM | CPSM | MWBM | CPSM | MWBM | ||
脱水 Dehydration | 70%乙醇 70% Ethanol | 60 | 1 | 120 | 6 | 120 | 6 |
85%乙醇 85% Ethanol | 60 | 1 | 120 | 6 | 120 | 6 | |
95%乙醇 95% Ethanol | 60 | 1 | 120 | 6 | 120 | 6 | |
100%乙醇 100% Ethanol | 60 | 1 | 120 | 6 | 120 | 6 | |
100%乙醇 100% Ethanol | 60 | 1 | 120 | 6 | 120 | 6 | |
透明 Clearing | 1/2乙醇 + 1/2二甲苯 1/2 Ethanol + 1/2 Xylene | 60 | 1 | 120 | 6 | 120 | 6 |
二甲苯 Xylene | 60 | 1 | 120 | 6 | 120 | 6 | |
二甲苯 Xylene | 60 | 1 | 120 | 6 | 120 | 6 | |
浸蜡 Dipping paraffin | 1/2二甲苯+石蜡 1/2 Xylene + Paraffin | 90 | 10 | 120 | 15 | 120 | 15 |
石蜡 Paraffin | 90 | 10 | 120 | 15 | 120 | 15 | |
石蜡 Paraffin | 90 | 10 | 120 | 15 | 120 | 15 | |
石蜡 Paraffin | — | 10 | — | 15 | — | 15 | |
总时长 Total time | 750 | 48 | 1 320 | 108 | 1 320 | 108 |
组织 Tissue | 方法 Method | 蜡带类型The wax bands types | 蜡带成带比率/% The ratio of wax band types | ||
---|---|---|---|---|---|
成带(片) Success of wax bands (piece) | 裂带(片) Split of wax bands (piece) | 不成带(片) Fail of wax bands (piece) | |||
叶片Leaf | CPSM | 30 | 0 | 0 | 100.0 |
MWBM | 30 | 0 | 0 | 100.0 | |
新梢Shoot | CPSM | 24 | 6 | 0 | 80.0 |
MWBM | 25 | 5 | 0 | 83.3 | |
花芽Flower bud | CPSM | 13 | 14 | 3 | 43.3 |
MWBM | 15 | 10 | 5 | 50.0 |
Table 2 Statistics of wax bands types between conventional paraffin sectioning(CPSM)and microwave and water bath(MWBM)in different pear tissues
组织 Tissue | 方法 Method | 蜡带类型The wax bands types | 蜡带成带比率/% The ratio of wax band types | ||
---|---|---|---|---|---|
成带(片) Success of wax bands (piece) | 裂带(片) Split of wax bands (piece) | 不成带(片) Fail of wax bands (piece) | |||
叶片Leaf | CPSM | 30 | 0 | 0 | 100.0 |
MWBM | 30 | 0 | 0 | 100.0 | |
新梢Shoot | CPSM | 24 | 6 | 0 | 80.0 |
MWBM | 25 | 5 | 0 | 83.3 | |
花芽Flower bud | CPSM | 13 | 14 | 3 | 43.3 |
MWBM | 15 | 10 | 5 | 50.0 |
方法 Method | 叶片 Leaf | 新梢 Shoot | 花芽 Flower bud | ||||||
---|---|---|---|---|---|---|---|---|---|
总片数 Total | 粘片 Section adhesion | 脱片 Slide detachment | 总片数 Total | 粘片 Section adhesion | 脱片 Section detachment | 总片数 Total | 粘片 Section adhesion | 脱片 Section detachment | |
水浴粘片 Water bath pasting | 100 | 100 | 0 | 100 | 100 | 0 | 100 | 100 | 0 |
明胶粘片剂 Gelatinglycerin adhesive | 100 | 100 | 0 | 100 | 90 | 10 | 100 | 88 | 12 |
Table 3 Statistics of the number of flakes removed by adhesive and water bath pasting methods
方法 Method | 叶片 Leaf | 新梢 Shoot | 花芽 Flower bud | ||||||
---|---|---|---|---|---|---|---|---|---|
总片数 Total | 粘片 Section adhesion | 脱片 Slide detachment | 总片数 Total | 粘片 Section adhesion | 脱片 Section detachment | 总片数 Total | 粘片 Section adhesion | 脱片 Section detachment | |
水浴粘片 Water bath pasting | 100 | 100 | 0 | 100 | 100 | 0 | 100 | 100 | 0 |
明胶粘片剂 Gelatinglycerin adhesive | 100 | 100 | 0 | 100 | 90 | 10 | 100 | 88 | 12 |
染色方式 Method | 步骤 Step | 时长/min Time |
---|---|---|
番红—固绿 Safranine-fast green | 透明剂(4 min)→1/2 乙醇 + 1/2透明剂(4 min)→100%乙醇(4 min)→95%乙醇(4 min)→85%乙醇(4 min)→70%乙醇(4 min)→纯水(4 min)→番红(240 min)→纯水(2 min)→70%乙醇(2 min)→85%乙醇(2 min)→95%乙醇(2 min)→固绿(1 min)→95%乙醇(2 min)→100%乙醇(2 min)→1/2乙醇+1/2 透明剂(2 min)→透明剂(2 min) Clearing Agent (4 min)→1/2 Ethanol +1/2 Clearing Agent (4 min)→100% Ethanol (4 min)→95% Ethanol (4 min)→85% Ethanol (4 min)→70% Ethanol (4 min)→Distilled water (4 min)→Safranine (240 min)→Distilled water (2 min)→70% Ethanol (2 min)→85% Ethanol (2 min)→95% Ethanol (2 min)→Fast green(1 min)→95% Ethanol (2 min)→100% Ethanol (2 min)→1/2 Ethanol +1/2 Clearing Agent (2 min)→Clearing Agent (2 min) | 285 |
固绿 Fast green | 透明剂(4 min)→1/2乙醇 + 1/2透明剂(4 min)→100%乙醇(4 min)→固绿(2 min)→95%乙醇(2 min)→100%乙醇(2 min)→1/2乙醇+1/2 透明剂(2 min)→透明剂(2 min) Clearing Agent (4 min)→1/2 Ethanol +1/2 Clearing Agent (4 min)→100% Ethanol (4 min)→Fast green (2 min)→95% Ethanol (2 min)→100% Ethanol (2 min)→1/2 Ethanol +1/2 Clearing Agent (2 min)→Clearing Agent (2 min) | 22 |
甲苯胺蓝 Toluidine blue | 透明剂(4 min)→1/2乙醇+1/2透明剂(4 min)→100%乙醇(4 min)→甲苯胺蓝(2 min)→95%乙醇(2 min)→100%乙醇(2 min)→1/2乙醇+1/2 透明剂(2 min)→透明剂(2 min) Clearing Agent (4 min)→1/2 Ethanol +1/2 Clearing Agent (4 min)→100% Ethanol (4 min)→Toluidine blue (2 min)→95% Ethanol (2 min)→100% Ethanol (2 min)→1/2 Ethanol +1/2 Clearing Agent (2 min)→Clearing Agent (2 min) | 22 |
苏木精—伊红 Hematoxylin-eosin | 透明剂(4 min)→1/2乙醇 + 1/2透明剂(4 min)→100%乙醇(4 min)→95%乙醇(4 min)→85%乙醇(4 min)→70%乙醇(4 min)→苏木精(10 min)→分化液(4 min)→返蓝液(2 min)→伊红(5 min)→70%乙醇(2 min)→85%乙醇(2 min)→95%乙醇(2 min)→100%乙醇(2 min)→1/2乙醇+1/2透明剂(2 min)→透明剂(2 min) Clearing Agent (4 min)→1/2 Ethanol +1/2 Clearing Agent (4 min)→100% Ethanol (4 min)→95% Ethanol (4 min)→85% Ethanol (4 min)→70% Ethanol (4 min)→Hematoxylin (10 min)→Differentiating Solution (4 min)→Bluing Solution (2 min)→Eosin (5 min)→70% Ethanol (2 min)→85% Ethanol (2 min)→95% Ethanol (2 min)→100% Ethanol (2 min)→1/2 Ethanol +1/2 Clearing Agent (2 min)→Clearing Agent (2 min) | 57 |
Table 4 Operation flow of four dyeing methods
染色方式 Method | 步骤 Step | 时长/min Time |
---|---|---|
番红—固绿 Safranine-fast green | 透明剂(4 min)→1/2 乙醇 + 1/2透明剂(4 min)→100%乙醇(4 min)→95%乙醇(4 min)→85%乙醇(4 min)→70%乙醇(4 min)→纯水(4 min)→番红(240 min)→纯水(2 min)→70%乙醇(2 min)→85%乙醇(2 min)→95%乙醇(2 min)→固绿(1 min)→95%乙醇(2 min)→100%乙醇(2 min)→1/2乙醇+1/2 透明剂(2 min)→透明剂(2 min) Clearing Agent (4 min)→1/2 Ethanol +1/2 Clearing Agent (4 min)→100% Ethanol (4 min)→95% Ethanol (4 min)→85% Ethanol (4 min)→70% Ethanol (4 min)→Distilled water (4 min)→Safranine (240 min)→Distilled water (2 min)→70% Ethanol (2 min)→85% Ethanol (2 min)→95% Ethanol (2 min)→Fast green(1 min)→95% Ethanol (2 min)→100% Ethanol (2 min)→1/2 Ethanol +1/2 Clearing Agent (2 min)→Clearing Agent (2 min) | 285 |
固绿 Fast green | 透明剂(4 min)→1/2乙醇 + 1/2透明剂(4 min)→100%乙醇(4 min)→固绿(2 min)→95%乙醇(2 min)→100%乙醇(2 min)→1/2乙醇+1/2 透明剂(2 min)→透明剂(2 min) Clearing Agent (4 min)→1/2 Ethanol +1/2 Clearing Agent (4 min)→100% Ethanol (4 min)→Fast green (2 min)→95% Ethanol (2 min)→100% Ethanol (2 min)→1/2 Ethanol +1/2 Clearing Agent (2 min)→Clearing Agent (2 min) | 22 |
甲苯胺蓝 Toluidine blue | 透明剂(4 min)→1/2乙醇+1/2透明剂(4 min)→100%乙醇(4 min)→甲苯胺蓝(2 min)→95%乙醇(2 min)→100%乙醇(2 min)→1/2乙醇+1/2 透明剂(2 min)→透明剂(2 min) Clearing Agent (4 min)→1/2 Ethanol +1/2 Clearing Agent (4 min)→100% Ethanol (4 min)→Toluidine blue (2 min)→95% Ethanol (2 min)→100% Ethanol (2 min)→1/2 Ethanol +1/2 Clearing Agent (2 min)→Clearing Agent (2 min) | 22 |
苏木精—伊红 Hematoxylin-eosin | 透明剂(4 min)→1/2乙醇 + 1/2透明剂(4 min)→100%乙醇(4 min)→95%乙醇(4 min)→85%乙醇(4 min)→70%乙醇(4 min)→苏木精(10 min)→分化液(4 min)→返蓝液(2 min)→伊红(5 min)→70%乙醇(2 min)→85%乙醇(2 min)→95%乙醇(2 min)→100%乙醇(2 min)→1/2乙醇+1/2透明剂(2 min)→透明剂(2 min) Clearing Agent (4 min)→1/2 Ethanol +1/2 Clearing Agent (4 min)→100% Ethanol (4 min)→95% Ethanol (4 min)→85% Ethanol (4 min)→70% Ethanol (4 min)→Hematoxylin (10 min)→Differentiating Solution (4 min)→Bluing Solution (2 min)→Eosin (5 min)→70% Ethanol (2 min)→85% Ethanol (2 min)→95% Ethanol (2 min)→100% Ethanol (2 min)→1/2 Ethanol +1/2 Clearing Agent (2 min)→Clearing Agent (2 min) | 57 |
步骤 Step | 试剂梯度 Reagent gradient | 时间/min Time | ||
---|---|---|---|---|
叶片 Leaf | 新梢 Shoot | 花芽 Flower bud | ||
固定Tissue fixation | FAA固定液 FAA fixative | 放置4 ℃冰箱保存 Store at 4 ℃ in a refrigerator | ||
脱水Dehydration | 70%乙醇 70% Ethanol | 1 | 6 | 6 |
85%乙醇85% Ethanol | 1 | 6 | 6 | |
95%乙醇95% Ethanol | 1 | 6 | 6 | |
100%乙醇100% Ethanol | 1 | 6 | 6 | |
100%乙醇100% Ethanol | 1 | 6 | 6 | |
透明Clearing | 1/2乙醇+1/2透明剂 1/2 Ethanol +1/2 Clearing agent | 1 | 6 | 6 |
透明剂 Clearing agent | 1 | 6 | 6 | |
透明剂 Clearing agent | 1 | 6 | 6 | |
浸蜡Dipping paraffin | 1/2透明剂 + 1/2石蜡 1/2 Clearing agent +1/2 Paraffin | 10 | 15 | 15 |
石蜡 Paraffin | 10 | 15 | 15 | |
石蜡 Paraffin | 10 | 15 | 15 | |
石蜡 Paraffin | 10 | 15 | 15 | |
固定、修蜡 Embedding and trimming paraffin | 常温条件下:蜡块固定120 min以上 Room temperature:Fix tissue blocks for 120 min 冷冻条件下:蜡块固定60 min左右 Freezing conditions:Fix frozen tissue blocks for 60 minute | |||
展、粘片 Unfolding and pasting | 水浴粘片 The water bath pasting method | 60 | ||
染色Dyeing | 透明剂 Clearing agent | 4 | ||
1/2透明剂 + 100%乙醇 1/2 Clearing agent + 1/2 Ethanol | 4 | |||
100%乙醇 100% Ethanol | 4 | |||
固绿染色剂 Fast green/甲苯胺蓝Toluidine blue | 2 | |||
95%乙醇 95% Ethanol | 2 | |||
100%乙醇 100% Ethanol | 2 | |||
1/2透明剂+100%乙醇 1/2 Ethanol +1/2 Clearing agent | 2 | |||
透明剂 Clearing agent | 2 | |||
制片 Sealing section | 盖玻片 Neutral gum | 中性树胶 Neutral mounting medium | ||
烘片 Drying section | 烘片 Slide drying | 30 | ||
中间过程中耗时时间 Time consuming in the intermediate process | 60 | |||
制片时间 Making time | 280 ~ 340 | 340 ~ 400 | 340 ~ 400 |
Table 5 Operation process of MWPS method for different pear tissue
步骤 Step | 试剂梯度 Reagent gradient | 时间/min Time | ||
---|---|---|---|---|
叶片 Leaf | 新梢 Shoot | 花芽 Flower bud | ||
固定Tissue fixation | FAA固定液 FAA fixative | 放置4 ℃冰箱保存 Store at 4 ℃ in a refrigerator | ||
脱水Dehydration | 70%乙醇 70% Ethanol | 1 | 6 | 6 |
85%乙醇85% Ethanol | 1 | 6 | 6 | |
95%乙醇95% Ethanol | 1 | 6 | 6 | |
100%乙醇100% Ethanol | 1 | 6 | 6 | |
100%乙醇100% Ethanol | 1 | 6 | 6 | |
透明Clearing | 1/2乙醇+1/2透明剂 1/2 Ethanol +1/2 Clearing agent | 1 | 6 | 6 |
透明剂 Clearing agent | 1 | 6 | 6 | |
透明剂 Clearing agent | 1 | 6 | 6 | |
浸蜡Dipping paraffin | 1/2透明剂 + 1/2石蜡 1/2 Clearing agent +1/2 Paraffin | 10 | 15 | 15 |
石蜡 Paraffin | 10 | 15 | 15 | |
石蜡 Paraffin | 10 | 15 | 15 | |
石蜡 Paraffin | 10 | 15 | 15 | |
固定、修蜡 Embedding and trimming paraffin | 常温条件下:蜡块固定120 min以上 Room temperature:Fix tissue blocks for 120 min 冷冻条件下:蜡块固定60 min左右 Freezing conditions:Fix frozen tissue blocks for 60 minute | |||
展、粘片 Unfolding and pasting | 水浴粘片 The water bath pasting method | 60 | ||
染色Dyeing | 透明剂 Clearing agent | 4 | ||
1/2透明剂 + 100%乙醇 1/2 Clearing agent + 1/2 Ethanol | 4 | |||
100%乙醇 100% Ethanol | 4 | |||
固绿染色剂 Fast green/甲苯胺蓝Toluidine blue | 2 | |||
95%乙醇 95% Ethanol | 2 | |||
100%乙醇 100% Ethanol | 2 | |||
1/2透明剂+100%乙醇 1/2 Ethanol +1/2 Clearing agent | 2 | |||
透明剂 Clearing agent | 2 | |||
制片 Sealing section | 盖玻片 Neutral gum | 中性树胶 Neutral mounting medium | ||
烘片 Drying section | 烘片 Slide drying | 30 | ||
中间过程中耗时时间 Time consuming in the intermediate process | 60 | |||
制片时间 Making time | 280 ~ 340 | 340 ~ 400 | 340 ~ 400 |
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