timm染色.docx

标本的制备：1.??开胸经左心室连续灌流：依次为生理盐水150ml(5-8min)，1%的硫化钠100ml，4%的多聚甲醛100ml，1%的硫化钠50ml。（我是用注射器灌的，硫化钠不能推的太快，否则脑组织膨胀的很厉害）2.??断头取脑，用4%的多聚甲醛在4℃固定过夜。-----有文献显示：取脑后沿中隔区切除包括海马在内的2-3mm脑组织后多聚甲醛固定。3.??在30%的蔗糖磷酸缓冲（PB）溶液中低温防护。------有文献显示：在30%的蔗糖溶液中浸泡24（或至48）h，直至组织块沉淀。一般要48小时才能沉底。4.??通过中隔海马（从后部到前囟2.8—4.0mm）恒温冷冻冠状切片，切片在PB（磷酸缓冲液）中连续收集。片厚为30微米。每隔100微米选一张片进行Timm染色。Timm组织化学染色：1.??切片贴附于多聚赖氨酸处理过的载玻片上。（-20°保存）2.??做的时候拿出来晾干（10分钟）。3.??把Timm显影剂倒入修复盒中，放在在26℃的水浴箱或恒温摇床里染色90-150min，染色必须在避光的条件下进行。4.??染色后，切片用蒸馏水冲洗几遍，可以尼式染色复染，也可不复染。5.??晾干，中性树脂封胶片。Timm液的配置：A:防护胶：30g阿拉伯胶搅拌溶解于60ml去离子水中（充分搅匀溶解）。静置两天，如果有杂质可用双层纱布滤过。胶不急用的话冷冻保存在塑料瓶里可以保存至少6个月。B：枸橼酸缓冲液：2.55枸橼酸?H2O加2.35枸橼酸钠?2H2O，加去离子水至10ml。C:还原剂：0.85g对苯二酚（氢醌）溶于15ml的去离子水。D:银离子供应：0.11g乳酸银（硝酸银）加入15ml的去离子水。乳酸银对光高敏感，因此此溶液应当避光。A B C溶液小心混合，D溶液在用之前立即加入。一次做八到十张片子比较合适。Timm Staining ProtocolFrom Nikki Sunnen, BCM 4/09Tissue Preparation Solutions1) Sulfide Perfusate??Sodium sulfide nonahydrate (Sigma #208043)??2.9g??Sodium phosphate (monobasic monohydrate)??3.0g??De-ionized water??????????250ml**mix until the solution turns clear. If you continue to mix past this point, it may turn yellow again, but should still be okay to use.2) 10% Neutral Buffered Formalin3) Post-Fixative??Gluteraldehyde (25%)??????????10ml??Dextrose????????????24g??De-ionized water??????????70mlTimm Stain Solution??The Timm stain solution is comprised of 4 different solutions. The recipes for these 4 comprising solutions are listed below, but the Timm stain solution is:??1) Gum Arabic??????????120ml??2) Citrate buffer??????????20ml??3) Hydroquinone??????????60ml??4) Silver nitrate??????????1ml1) Gum Arabic??Gum arabic????????????500g??De-ionized water??????????1000ml***This will take 3-4 days to dissolve! Prepare once, then aliquot into 50ml tubes and freeze at -20°C.***2) Citrate Buffer??Citric acid????????????5.1g??Sodium citrate????????????4.7g??De-ionized water??????????to 20ml3) Hydroquinone Developer??Hydroquinone????????????3.4g??De-ionized water??????????to 60ml**takes some time to dissolve, so use