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This protocol aims to introduce methods for sacrificing rats by transcardial perfusion and extracting the brain, and introduce methods for staining the rat brain tissue with c-Fos and Arc antibodies. Please note the expression of the proteins is very sensitive to behavioral paradigm that triggers neural activity.

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c-Fos and Arc Immunohistochemistry on Rat Cerebellum
基于大鼠小脑的c-Fos 及Arc免疫组织化学

神经科学 > 行为神经科学 > 学习和记忆
作者: Soyun Kim
Soyun KimAffiliation: Neuroscience Program, University of Southern California, Los Angeles, USA
For correspondence: soyunkimucsd@gmail.com
Bio-protocol author page: a45
Vol 2, Iss 10, 5/20/2012, 10757 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.191

[Abstract] This protocol aims to introduce methods for sacrificing rats by transcardial perfusion and extracting the brain, and introduce methods for staining the rat brain tissue with c-Fos and Arc antibodies. Please note the expression of the proteins is very sensitive to behavioral paradigm that triggers neural activity.

[Abstract] 本实验方案目的为介绍穿心灌注死亡大鼠和采集大脑的方法c,和介绍用-Fos和Arc抗体染色大鼠脑组织。请注意这些蛋白的表达与行为范式激发的神经活动相关。

Materials and Reagents

  1. c-Fos (raised in mouse, 1:1,000 dilution) (Santa Cruz Biotechnology, catalog number: sc-8074 )
  2. Arc (raised in rabbit, 1:1,000 dilution) (Synaptic Systems, catalog number: 156002 )
  3. c-Fos IHC, Alexa antimouse Fluor 488 (Life Technologies, Invitrogen™, catalog number: 51663A )
  4. Arc IHC, Cy3-conjugated donkey anti-rabbit secondary antibody with 1:500 dilution (Jackson Laboratory, catalog number: 88069 )
  5. Euthasol (Virbac, catalog number: 710101 )
  6. Vectashield (Vector laboratory, catalog number: H-1000 )
  7. PFA powder
  8. 0.1 M PBS (pH 7.4)
  9. TritonX-100
  10. NaHPO4
  11. NaH2PO4
  12. Paraformaldehyde (PFA)
  13. NaOH
  14. NaCl
  15. Sucrose
  16. Eythlen glycol (RNAse free) (Sigma-Aldrich, catalog number: E9129 )
  17. Glycerol (RNase free)
  18. Phosphate buffer (PB) (0.2 M stock, pH 7.4) (Solution 1) (see Recipes)
  19. 4% Paraformaldehyde (PFA) for transcardial perfusion (Solution 2) (see Recipes)
  20. 0.9% Saline (Solution 3) (see Recipes)
  21. 30% sucrose in 4% PFA used as cryoprotectant (Solution 4) (see Recipes)
  22. Phosphate buffer saline (PBS) (Solution 5) (see Recipes)
  23. Cryoprotectant (Solution 6) (see Recipes)
  24. Normal donkey serum containing 0.25% Triton-100 (see Recipes)
  25. Primary Antibody (see Recipes)
  26. Secondary Antibody (see Recipes)

Equipment

  1. Fume hood
  2. Water tab
  3. Rib cage
  4. Scissors
  5. Forceps
  6. Clippers
  7. Cannula
  8. Vial
  9. Parafilm
  10. Refrigerator
  11. Tinfoil
  12. Coverslip

Procedure

  1. Perfusion
    1. To prepare solution, please refer to recipes. Let the solution flow by gravity (e.g. hanging bottles on top of the fume hood). About 250 ml/ rat is required.
    2. Prepare all equipment needed (scissors, forceps, clippers to hold hands/feet…etc).
      Note: Better to work close to the water tab (for rinsing out the coagulated blood during perfusion).
    3. Anesthetize the animal (overdosed with pentobarbital, 0.5 ml-1 ml/rat depending on weight) at least 30 min after behavioral treatment (this time allows protein synthesis).
    4. Pinch the toes to check the animal is fully asleep.
    5. Locate the sternum and poke into it using a scissor.
    6. Cut the chest cavity in V shape above the liver.
    7. Hold the sternum with a clip, and flip over the head to open the rib cage.
      *(optional) pinching the aorta behind the liver will help blood circulation into the brain. After pinching the aorta, let the forelimbs/hindlimbs relax (if clamped).
    8. Insert a cannula connected to the solution bottle into the apex of the heart near the left ventricle. Hold the cannula so that it is slightly pointing towards the right atrium.
    9. Start the saline flow.
    10. Immediately, cut (~0.3 cm) the right atrium.
    11. Rinse as necessary to remove the coagulated blood. You can try scraping out the debris using a forcep.
    12. Once the solution turns transparent (no more red blood cells), let the PFA flow (if you need immediate fixation, fix with PFA. No need to wait for the solution turning transparent).
    13. Leave until the solution is used up (could be longer than 20 min depending on the flow rate, weight…etc). If you see solution drops coming out of nostrils, it is a good sign.
    14. Extract the brain out of the skull carefully.
    15. Prepare a vial (tube) for each brain filled with cryoprotectant (solution 6).
    16. Store until the brain sinks to the bottom. Replacing the solution every day or so is recommended.
    17. For longer storage, embed in the OCT compound and store at -80 °C until use.
      Note: If you choose to cut the coronal sections using cryostat:
      Cut the tissue at 30 μm (could be in the range of 25-50 μm).
      Free-floating method: collect the sections in 24 well. Store in PBS (pH 7.4, 0.1 M) under 4 °C. Work immediately, if possible. Otherwise, store in cryoprotectant (solution 6) under -20 °C.

  2. Immunohistochemistry (IHC, Free-floating method)
    Note: If sections are mounted onto a slide immediately after cut, about 0.3 ml working solution is needed per slide. This method will help you save some antibodies. The disadvantage is that antibody may not be penetrating the tissue as effectively as in free-floating method. Also, slides might be easily dried out.
    For free-floating method, ~0.5 ml is required per well. For 30 μM-thick sections, about 7-8 sections can undergo identical treatment within a well. Use a dropper with a very thin tip to drain out solutions. Make sure the sections are not damaged during washing/solution change.
    1. Start with washing in PBS (3 times, 5 min each).
    2. Block the tissue with normal donkey serum containing 0.25% Triton-100. 1 h at room temperature (RT).
    3. Prepare primary antibody in the blocking solution.
      *Vortex briefly for diluting an antibody.
      Primary antibody incubation carried out over night at 4 °C. Wrapping the well with parafilm will prevent dehydration. For immediate results, one could incubate at RT for ~2-3 h. Overnight incubation is recommended for sufficient antibody incubation.
    4. Take the well out of refrigerator and let it sit at RT for ~1 h.
    5. Wash with PBS (3 times, 5 min each).
    6. Secondary antibody incubation under RT 1-2 h.
    7. Following incubation, wash with PB (NOT PBS!) for 3 times, 5 min each.
    8. Mount the sections carefully onto slides. Air dry while covered on top (with tinfoil, or any type of lid that prevents light).
    9. Apply a drop of Vectashield (Vector laboratory) before coverslipping.
    10. For storage, keep at -80 °C.

Recipes

  1. Solution 1: Phosphate buffer (PB) (0.2 M stock, pH 7.4)
    Monobasic NaP
    ddH2O (double-distilled) 250 ml
    NaH2PO4 6.9 g
    Dibasic NaP
    ddH2O 1 L
    NaHPO4 28.4 g
    In ~100 ml of Monobasic NaP, start adding Dibasic NaP. Measure pH while adding Dibasic (~600-800 ml may be needed).
    Dilute this 0.2 M stock solution by 1/2 using ddH2O.

  2. Solution 2: 4% Paraformaldehyde (PFA) for transcardial perfusion (make fresh for each use. Storage of up to 3-4 days okay)
    To make 1 L of PFA:
    Heat 900 ml ddH2O to 60 °C (Do NOT exceed 70 °C)
    Add 40 g of PFA powder in the fume hood. Stir ~5 min
    Add 10 drops of 2N NaOH until the solution gets clear (could take a couple of min). Keep stirring
    Remove from heat and add 100 ml of 0.1 M PB (Solution1)
    Filter and store at 4 °C overnight. Filtration is crucial; otherwise, the PFA debris will clog up the blood vessel
    Adjust pH at 4 °C (standardization required under this temperature prior to measurement)
  3. Solution 3: 0.9% Saline (~500 ml required/ rat brain)
    NaCl 9 g/L of ddH2O
    (Optional) add heparin for better circulation. 200 units/L of working solution recommended
  4. Solution 4: 30% sucrose in 4% PFA used as cryoprotectant
    Store refrigerated.
    Add 30% (g) sucrose to freshly made 4% PFA (Solution 2).
  5. Solution 5: Phosphate buffer saline (PBS)
    0.01 M sodium phosphate in 0.9% saline
    To make 5 L
    Add 45 g NaCl, 250 ml of 0.2 M PB (Solution1) into 4.5 L of ddH2O.
    (Optional, but could be very helpful for perfusion) Add heparin (200 units/L working solution) to prevent blood clotting.
  6. Solution 6. Cryoprotectant (for saving cut sections over a long term)
    50% 0.05 M PB (dilute 0.2 M PB stock)
    30% eythlen glycol (RNAse free)
    20% glycerol (RNAse free)
  7. Normal donkey serum containing 0.25% Triton-100
    For 1 ml aliquot,
    25 μl of 10% Triton
    875 μl of PBS
    100 μl normal donkey serum
    * Do not exceed 1% triton for brain tissues.
  8. Primary Antibody
    c-Fos
    Arc
    1 μl in 1,000 μl blocking solution
  9. Secondary Antibody
    c-Fos IHC, Alexa antimouse Fluor 488 in 1:500 dilution.
    Arc IHC, Cy3-conjugated donkey anti-rabbit secondary antibody with 1:500 dilution.
    Dilute antibodies in PBS (NOT blocking solution).

Acknowledgments

This protocol was adapted from Kim and Thompson (2011).

References

  1. Kim, S. and Thompson, R. F. (2011). c-Fos, Arc, and stargazin expression in rat eyeblink conditioning. Behav Neurosci 125(1): 117-123.

材料和试剂

          

1.        c-Fos (小鼠产生, Santa Cruz, sc-8074, 1:1000稀释度)

2.        Arc (兔子产生, Synaptic Systems, Cat# 156 002, 1:1000 稀释度)

3.        c-Fos IHC, Alexa antimouse Fluor 488 (Invitrogen, Lot 51663A)

4.        Arc IHC, Cy3-接合的驴抗兔二抗 1:500 稀释度(Jackson Laboratory, Lot 88069)

5.        Euthasol (Virbac, 710101)

6.        PBS

7.        普通驴血清

8.        Triton-100

9.        PB

10.    Vectashield (Vector laboratory, H-1000)

11.    一价NaP

12.    二价 NaP

13.    ddH2O

14.    NaHPO4

15.    低聚甲醛(PFA)

16.    NaOH

17.    NaCl

18.    蔗糖

19.    乙二醇 (RNAse free) (Sigma #E 9129)

 

仪器

 

1.        剪刀

2.        镊子

3.        钳子

4.        套管

5.        管形瓶

6.        封口膜

7.        冰箱

8.        锡箔

9.        盖玻片

 

步骤

 

1.        灌流

1)       准备配置方法中的溶液. 让溶液在重力作用下流动 (e.g. 将瓶子绑在通风厨的顶部).大约需要250 ml/ 大鼠。

2)       准备所有所需要的仪器 (剪刀,镊子, 钳子住手/…etc).

注意: 最好在水龙头旁边操作 (在灌注时冲刷凝固的血液).

3)       麻醉动物(用过量的戊巴比妥,0.5 ml~1 ml/大鼠,依据为大鼠的体重。) 在处理后至少要30min (这个时间使蛋白合成).

4)       捏脚趾检查动物是否已经完全麻醉。.

5)       确定胸骨的位置并且用剪刀插入。

6)       在肝脏上面的胸腔切开一个V字形的切口。

7)       用一个夹子夹住胸骨,翻动头部打开肋架

*(可选择)挤压肝后面的主动脉将帮助血液循环到脑部,然后使得前肢/后肢放松(如果固定着)。

8)       插入个连着溶液瓶子的插管到左心室旁边的骶骨尖. 握着管子使其轻轻的顶住右心房。

9)       使盐水流动。.

10)   迅速的,切开 (~0.3 cm) 右心房。.

11)   如果需要冲洗凝固的血液,你可以用钳状骨针挂掉残余组织。.

12)   一旦溶液变得透明 (不再有血细胞), 使 PFA 流动 (如果你需要迅速的固定,用PFA固定. 不需要等到溶液变得透明).

13)   等到溶液用光 (取决于流速和重量等有关,大概需要比20min以上).如果你看到溶液从鼻子孔流出,是一个溶液用光的标志。

14)   小心的取下头盖骨采集大脑组织。.

15)   为每一个脑组织准备一个加上抗冻溶剂(溶液 6).的管子。 

16)   直到脑组织下沉到底部然后保存,建议每天都要换溶液。.

17)   如果要长期储存,包埋到OCT化合物中放于-80 °C保存。

18)   注意:如果你选择用低温恒温器切冠状缝切面。

将组织切成 30 μm (尽可能在 25-50 μm).

自由浮动方法:收集片段于 24 管中,4 °C储存于PBS(pH 7.4, 0.1 M) 如果可能时间尽量可能的短,否则,在-20 °C.储存于抗冻剂。

2.        免疫组化 (IHC, 自由浮动法)

注意:如果切除后,片段迅速包埋到在载玻片上,每个载玻片需要大约0.3ml工作溶液。这个方法可以帮助节省抗体。缺点是抗体可能不能有限的穿透组织,载玻片也很容易干掉。

对于自由浮动法:每一个管子要用 ~0.5 ml。对于. 30 μM厚的切片,大约7-8 切片可以 在同一管子中得到恒定的处理。 用带有非常细头的滴管去吸去溶液。确保片断在洗涤或者换液的时候不会受到损伤。

1)        开始用PBS洗涤。 (3 , 每次5 mins).

2)        用含有 0.25% Triton-100. 的驴血清封闭组织,室温1个小时。.

3)        在封闭液中准备抗。.

*稀释抗体时轻轻的涡旋震荡。

4 °C抗封闭过夜,用封口膜封住防止脱水。如果想迅速得到结果,可以在室温下孵育23个小时,建议过夜孵育,这样可以孵育的更加的充分。

4)        从冰箱中拿出管子,置于室温下 ~1hr

5)        PBS洗涤。 (3 , 每次5 mins).

6)        二抗室温下孵育 1-2 hrs.

7)        孵育后,用PB洗涤 (不是PBS!) 3次,每次5分钟。

8)        制作切片,在上面盖上东西后晾干(可以用锡箔纸,或者其它避光的物质)

9)        盖上盖玻片之前加上一滴Vectashield (Vector laboratory)

10)    置于 -80 °C保存。

 

配置方法

 

1.        溶液1: 磷酸盐缓冲液 (PB) (0.2 M stock, pH 7.4)


一价NaP

ddH2O (重蒸馏) 250 ml

NaH2PO4 6.9 g

二价 NaP

ddH2O 1 L

NaHPO4 28.4 g

 ~100 ml 一价NaP, 加入二价 NaP. 检测PH(可能需要~600-800 ml).

ddH2O稀释两倍0.2 M 的储存液

 

2.        溶液 2: 穿心灌注准备4%的低聚甲醛 (PFA)(每次使用最好配置新鲜的,可以存最多3-4.)

配置1LPFA,

加热 900 ml ddH2O60 °C (Do NOT 超过 70 °C).

在通风中加40g PFA 粉末 搅拌 ~5 mins.

10 2N NaOH直到溶液变得澄清(可能要几分钟时间). 保持搅拌。

停止加热加100 ml0.1 M PB Solution1).

过滤,在 4 °C 保存过夜,过滤比较困难,因为PFA残渣可能会堵塞血管。

4 °CpH (Standardization required under this temperature prior to measurement).

3.        Solution 3: 0.9% 盐水 (需要~500 ml /大鼠脑组织)

NaCl 9g/1L of ddH2O.

(可选择性) 为了更好地循环加些肝素,建议200 units/1 L 工作浓度。.

4.        Solution 4. 30%蔗糖在4% PFA作为冷冻干燥剂,冷冻保存。

30% (g) 蔗糖  4% PFA (Solution 2).

5.        Solution 5.磷酸缓冲液(PBS) (0.9% 0.01 M 磷酸钠)

配置 5 L,

 45 g NaCl, 250 ml of 0.2M PB (Solution1)4.5 L ddH2O

(选择性,可能对灌满非常有用) 加肝素(200 units/L 工作浓度) 防止血液凝固。.

6.        Solution 6.冷冻保护剂(为了长时间的储存切片)

50% 0.05 M PB (稀释0.2 M PB stock)

30%乙二醇(RNAse free)

20% 甘油 (RNAse free)

7.        普通驴血清含有0.25% Triton-100

1 ml 体积,

25 μl 10% Triton

875 μl PBS

100 μl 普通驴血清

*对于脑组织不要超过 1% triton

8.        

c-Fos

Arc

1 μl 加入到1000 μl封闭液中。

9.        二抗

c-Fos IHC, Alexa antimouse Fluor 488 稀释500倍。

Arc IHC, Cy3-接合 驴抗兔二抗500倍稀释。.

PBS稀释(不是封闭液).

 

References

 

1.         Kim S., Thompson R.F. (2011). c-Fos, Arc, and stargazin expression in rat eyeblink conditioning. Behavioral Neuroscience 125(1): 117-23. 

 

 

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How to cite this protocol: Kim, S. (2012). c-Fos and Arc Immunohistochemistry on Rat Cerebellum. Bio-protocol 2(10): e191. DOI: 10.21769/BioProtoc.191; Full Text



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