FJB染色中英文对照3.31

Amino-Cupric-Silver T echnique Materials
材料
Reagents 试剂
Stock solutions
储存液
Dissolve the gelatin completely on a hot plate, allow it to cool but not solidify, and then add the alcohol very slowly, with continuous mixing, to avoid precipitating the gelatin. This mounting solution can be stored for a long time at 4℃, at which point it is a cloudy gel. Before each use, put approximately 10mL of this solution in a beaker and warm it enough to redissolve and clarify it.
取明胶2g，加入去离子水100ml，在电炉上加热，使其充分溶解，稍微冷却，然后缓慢加入无水乙醇100ml，继续搅拌混均，避免加入过程中使明胶产生沉淀。

混均后，置于4℃下保存。

使用前取10ml 此溶液置于烧杯中，温浴使其溶解，澄清。

7. Cacodylate Buffer Solution
7. 二甲胂酸缓冲液
dH2O 1000 ml
纯水1000ml
NaCl 8 g
氯化钠8 g
Dextrose 4 g
右旋葡萄糖 4 g
Sucrose 8 g
蔗糖8 g
CaCl2 0.23 g
氯化钙0.23 g
Cacodylate 0.34 g
二甲胂酸0.34 g
Begin with 800ml dH2O of water and heat to 60-65°C (~104F) on a heater/ magnetic stirrer. Add the 8g NaCl. Create a deep vortex using a magnetic stirrer bar. Gradually sprinkle the finely Dextrose onto the vortex surface. Add the Suc rose. When dissolved, Add CaCl2. When dissolved, Add Cacodylate. Allow to cool to room temperature. Bring the total volume to 1000 ml with dH2O. Adjust the pH to 7.2 - 7.4 with concentrated HCl dropwise (ca. 15-20 drops).
量取800 mL纯水，置于电磁搅拌器中，加热至60-65℃。

加入蔗糖，溶液进行搅拌，并使其产生一个深深的漩涡，溶解后，往漩涡表面缓慢加入8g NaCl.待其溶解后，加入4g右旋葡萄糖。

溶解后，加入8g 蔗糖。

溶解后，加入0.23氯化钙。

溶解后，加入二甲胂酸，冷却至室温，加水至1000mL。

用浓盐酸调pH 至7.2-7.4（约15-20滴）。

8. Perfusion Fix:
8. 灌注液
dH2O 1000ml
纯水1000ml
Paraformaldehyde 40 g
多聚甲醛40 g
Sucrose 40 g
蔗糖40 g
Cacodylate 14.34 g
二甲胂酸14.34 g
Begin with 800ml dH2O of water and heat to 60-65°C (~104F) on a heater/ magnetic stirrer. Add the sucrose. Create a deep vortex using a magnetic stirrer bar.
Gradually sprinkle the finely powdered paraformaldehyde onto the vortex surface. The solution will be quite milky. Add the Sodium Cacodylate. The solution will clear considerably but still may be turbid and will require filtration with a qualitative paper. Allow to cool to room temperature. Bring the total volume to 1000 ml with dH2O. Adjust the pH to 7.2 - 7.4 with concentrated HCl dropwise (ca. 15-20 drops).
量取800 mL纯水，置于电磁搅拌器中，加热至60-65℃。

加入蔗糖，溶液进行搅拌，并使其产生一个深深的漩涡，溶解后，往漩涡表面缓慢加入多聚甲醛粉末，此时溶液呈乳白色。

加入二甲胂酸，此时溶液变澄清，底部略微产生沉淀，用滤纸过滤即可。

冷却至室温，加水至1000mL，用浓盐酸调pH至7.2-7.4（约15-20滴）。

Methods
Tissue preparation
组织制备
1. Fix rodent brains or spinal cord by intracardiac perfusion.
行心内灌注对大鼠脑和脊髓进行固定
2. 后固定
After perfusion fixation, remove the brains or spinal cords and store them in the same fixative for at least 24 h at 4℃. For faster perfusion, fix in 8% paraformaldehyde at room temperature overnight, with gentle agitation on shaker.
灌注固定后，取大脑脊髓，于相同的固定液中4℃下至少保存24h。

快速固定，可在室温下于8%多聚甲醛溶液中固定，用摇床慢摇过夜即可。

3. dehydrate
3. 脱水
Put brain into 0.1M Cacodylate buffer containing 15% Sucrose，until it sink the bottom.
将组织置于15％蔗糖二甲胂酸缓冲液内，直至沉入瓶底
Put brain into 0.1M Cacodylate buffer containing 25% Sucrose，until it sink the bottom.
将组织置于25％蔗糖二甲胂酸缓冲液内，直至沉入瓶底
4. prepare 50-um thick frozen section。

Silver staining works also on 40 um to 100 um thick brain, spinal cord, or retina sections. Store tissue sections in 4% paraformaldehyde at 4℃for up to 6 months.
4. 行冰冻切片，作一50um厚度的切片。

同样可在40-100um之间厚度的脑组织，脊髓组织，视网膜切片进行Silver staining。

切片可在4℃下，于4%的多聚甲醛溶液中保存至6个月。

Staining
染色
结果：正常组织颜色为：草黄色；溃变纤维颜色为：黑色。