Golgi-Cox染色(中文版)

1、golgi-cox染色step1：配置 golgi-cox溶液a：5%重铬酸钾。 10g重铬酸钾 +200ml 蒸馏水b：5%氯化汞。 10g 氯化汞 +200ml蒸馏水（于电炉上搅拌溶解）c：5%铬酸钾。 8g铬酸钾 +160ml 蒸馏水将 ab混合，c中加入 400ml 蒸馏水后，将 ab倒于 c中。避光于 26烘箱储存 5 天备用。step2：用塑料注射器从中间吸取gc溶液置于 50ml 试管中，避免吸到溶液底部和表面的红色沉淀物，每管加液量为该管预收集组织的4-5 倍体积。step3： 用 0.9%的生理盐水对实验动物进行心脏灌流，针插右心室，剪开左心耳。取大脑置于 gc溶液中， 2

2、天后换新 gc溶液，共避光处理14 天。step4：用 1000ml 蒸馏水溶解 300g 蔗糖，电炉加热溶解， 冷却待用。 取出大脑，用吸水纸吸取表面液体， 置于蔗糖溶液中 （每管加液量为该管预收集组织的 4-5 倍体积） 。于 4冰箱中，待组织下沉即可切片。 （一般不超过24h）step5：振动切片。切片槽中为6%的蔗糖溶液（ 100ml 蒸馏水溶解 6g 蔗糖） 。切片厚度 200m，贴片，稍吸干边缘水分，置于湿盒中4湿润保存，切好的片子放置过夜后处理（立即处理掉片较严重）。step6：染色1. 蒸馏水1min 2. 氨水60min 3. 蒸馏水1min 4. 柯达 fix 30min

3、5. 蒸馏水1min 6. 50%酒精 1min 7. 70%酒精 1min 8. 95%酒精 1min 9. 100%酒精 8min 10. 100%酒精 8min 11.cax 15min 中性树胶封片golgi-cox staining protocol for neurons and processes contributed by tracey wheeler george mason university novaultra special stain kits(tracey s way there are others as well, so dont be nervous if

4、 you come across them!) step 1: prepare the golgi-cox solution solution a: 5% potassium dichromate in distilled h2o 200ml distilled h2o + 10 grams potassium dichromate (mix in a glass beaker using a glass rod best to do under fume hood) solution b: 5% mercuric chloride (sublimate) in distilled h2o 2

5、00ml distilled h2o + 10 grams mercuric chloride (mix in a glass beaker using a glass rod) (mix solution on top of hotplate (on 5), stirring until dissolved) (must be done under fume hood) solution c: 5% solution of potassium chromate in distilled h2o 160ml distilled h2o + 8 grams potassium chromate

6、(mix in a glass beaker using a glass rod best to do under fume hood) mix solution a and solution b into a 500ml glass beaker. mix solution c and 400ml of distilled h2o into a 1,000ml + glass beaker. slowly pour the ab solution into the c solution while stirring continuously with a glass rod. store i

7、n a glass stoppered bottle for 5 days in the dark. note: you can easily manipulate the quantity of solution. just make sure to follow these ratios5 volume parts of 5% potassium dichromate solution 5 volume parts of 5% mercuric chloride solution 4 volume parts of 5% potassium chromate solution 10 vol

8、ume parts of h20 (to add to pc solution) step 2: transfer golgi-cox solution into small glass bottles. use a plastic syringe to remove the gc solution from the large glass bottle(s). be sure to avoid the reddish precipitate that formed on the top and bottom of the bottle. glass bottles should be fil

9、led about ? full (to save room for 1 rat brain). step 3: sacrifice rats using saline perfusion technique. deeply anaesthetize the animal. place on an empty breeder box with wire top. (to allow blood to drain into box) open chest cavity to expose heart insert 60 ml syringe filled with 9% saline into

10、bottom right chamber of the heart. (this would be the animals left chamber)using scissors, cut the bottom left chamber of the heart open. (this would be the animals right chamber)begin to slowly push saline through the animal s system until the blood leaving the left chamber is clear. (this may take

11、 3 syringes of saline.) when fluids are clear, decapitate and remove brain. drop whole brain into prepared bottle(s) of golgi cox solution. place in the dark for 14 days, refresh solution after 2 days. step 4: transfer brains into sucrose solution. mix 300 grams of sucrose into 1000ml of distilled h

12、2o. place beaker over hotplate and stir (using stir bar) until dissolved. cool in refrigerator. (once cool, ready to use.) empty gc solution from jar and place brain on chem. wipe paper. slightly blot. rinse jar in distilled h2o, and refill with sucrose solution ? full. (in order to save room for br

13、ain.) place brain in jar with sucrose solution. brain will float. place jar(s) in refrigerator. (once brains sink, they are ready to be sectioned.) (brains should be sectioned within 2 months of transfer into sucrose.) step 5: section using a vibratome. prepare razor blade by immersion in xylene for

14、 5 minutes to remove any traces of oil. (this should be done under the fume hood.) wipe blade dry. prepare a 6% sucrose solution. (6 grams sucrose in 100ml distilled h2o.) mix well and make sure it is at room temperature or below before using. fill the vibratome reservoir with the 6% sucrose solutio

15、n until the blade is covered. mount brain section (up to ? a full brain) onto vibratome platform using superglue. (make sure tissue is adhered well before sectioning, 5-7 ) or more.) insert platform (with adhered brain section) into reservoir. set the vibratome speed and amplitude around the midpoin

16、t for sectioning (adjust as necessary for your specific machine and comfort level.) section at 200 micro meters or desired thickness. (sections over 400 may be difficult to analyze.) using a small paintbrush coax the section onto a gelatinized slide. cover tissue with parafilm. place slide on flat s

17、urface covered with bibulous paper. place another sheet of bibulous paper over paraflim. place your palm over the section an press down slightly, being careful not alter movement. (your goal is to press the section into the gelatin on the slide so it will adhere to slide during staining.) remove bib

18、ulous paper and place in humidity chamber. note: we used a water sleeved incubator. in this apparatus it is necessary to keep the parafilm on the section. we also placed the slides on plastic trays which also held capfuls of water to be sure the slides did not dry out. do not keep the slides in stor

19、age (humidity chamber or water incubator) for more than 4 days.) step 6: staining prepare fresh solutions (enough to cover all slides): distilled h2o (3 total) ammonium hydroxide (1 total - keep under fume hood!) kodak fix (1 total keep under fume hood, mix in dark) 50% alcohol (1 total) 70% alcohol

20、 (1 total) 95% alcohol (1 total) 100% alcohol (3 total) cxa solution (1 total keep under fume hood!) kodak fix solution: prepare all ingredients in beakers mix in order: (do not mix in light) 1010 ml distilled h2o (add) 251 ml kodak fix solution a (add) 28 ml kodak fix solution b (add) 2020 ml disti

21、lled h2o (you can manipulate amounts, just use one half or 1/3 each) cxa solution: 1000 ml chloroform 1000 ml xylene 1000 ml 100% alcohol (you can manipulate amounts, just use one half or 1/3 each) remove parafilm from slides if necessary and place in slide rack. dip according to process below: 1. d

22、istilled h2o for 1 minute 2. ammonium hydroxide for 30 minutes (in the dark) (using a darkroom light mix the kodak fix solution now.) 3. distilled h2o for 1 minute (best to just keep lights off) 4. kodak fix solution for 30 minutes (in the dark) 5. distilled h2o for 1 minute (once in h2o you can tur

23、n on lights) 6. 50% alcohol 1 minute 7. 70% alcohol 1 minute 8. 95% alcohol 1 minute 9. 100% alcohol 5 minutes 10. 100% alcohol 5 minutes 11. 100% alcohol 5 minutes 12. cxa 15 minutes (keep under fume hood) (keep slides in cxa under fume hood while cover slipping, pull one slide out at a time.) note: change gloves often they will disintegrate in cxa. step 7: coverslip with permount and lie out to dry. if possible all cover slipping should be done under f