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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, 11433 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(在小鼠中生长,1:1000稀释)(Santa Cruz Biotechnology,目录号:sc-8074)
  2. 弧(在兔中生长,1:1000稀释)(Synaptic Systems,目录号:156002)
  3. c-Fos IHC,Alexa antimouse Fluor 488(Life Technologies,Invitrogen TM,目录号:51663A)
  4. Arc IHC,1:350稀释的Cy3-缀合的驴抗兔二抗(Jackson Laboratory,目录号:88069)
  5. Euthasol(Virbac,目录号:710101)
  6. Vectashield(Vector laboratory,目录号:H-1000)
  7. PFA粉末
  8. 0.1M PBS(pH7.4)
  9. TritonX-100
  10. NaHPO
  11. NaH 2 PO 4 sub
  12. 多聚甲醛(PFA)
  13. NaOH
  14. NaCl
  15. 蔗糖
  16. 乙二醇(无RNA酶)(Sigma-Aldrich,目录号:E9129)
  17. 甘油(不含RNase)
  18. 磷酸盐缓冲液(PB)(0.2M储备液,pH 7.4)(溶液1)(参见配方)
  19. 4%用于经心脏灌注的多聚甲醛(PFA)(溶液2)(参见配方)
  20. 0.9%盐水(解决方案3)(参见配方)
  21. 30%蔗糖在4%PFA中用作冷冻保护剂(溶液4)(参见配方)
  22. 磷酸盐缓冲盐水(PBS)(溶液5)(参见配方)
  23. 冷冻保护剂(解决方案6)(参见配方)
  24. 含有0.25%Triton-100的正常驴血清(见Recipes)
  25. 一抗(见配方)
  26. 次级抗体(参见配方)

设备

  1. 通风橱
  2. 水标签
  3. 肋骨架
  4. 剪刀
  5. 镊子
  6. 快船
  7. 插管
  8. 小瓶
  9. parafilm
  10. 冰箱
  11. 锡箔
  12. 盖玻片

程序

  1. 灌注
    1. 要准备解决方案,请参阅配方。 让溶液通过重力流动(例如,在通风橱顶上悬挂瓶子)。 需要约250ml /鼠。
    2. 准备所需的所有设备(剪刀,镊子,剪刀抓住手/脚...等) 注意:最好在靠近水位的地方工作(在灌注期间冲洗凝结的血液)。
    3. 麻醉动物(过量的戊巴比妥,0.5毫升-1毫升/大鼠,取决于体重)行为治疗后至少30分钟(这一次允许蛋白质合成)。
    4. 捏趾脚以检查动物是否完全睡着了。
    5. 找到胸骨,用剪刀戳它
    6. 切割肝上方V形的胸腔。
    7. 用夹子握住胸骨,翻转头部打开肋骨。
      *(可选)捏在肝脏后面的主动脉将有助于血液循环进入大脑。夹紧主动脉后,让前肢/后肢放松(如果夹紧)。
    8. 将连接到溶液瓶的插管插入左心室附近心脏的心尖部。握住插管,使其稍微指向右心房。
    9. 开始盐水流。
    10. 立即切开(〜0.3厘米)右心房。
    11. 必要时冲洗以除去凝结的血液。你可以尝试使用镊子刮掉碎屑。
    12. 一旦溶液变得透明(没有更多的红细胞),让PFA流动(如果你需要立即固定,用PFA固定,不需要等待溶液变透明)。
    13. 离开直到溶液用完(根据流速,重量等,可以超过20分钟)。如果你看到溶液从鼻孔流出,这是一个好兆头
    14. 仔细地从头骨中提取脑。
    15. 为填充有冷冻保护剂(溶液6)的每个大脑准备一个小瓶(管)
    16. 存储直到大脑下沉到底部。 建议每天更换溶液。
    17. 对于更长的存储,嵌入OCT化合物并存储在-80°C直到使用 注意:如果您选择使用低温恒温器切割冠状切片:
      将组织切成30微米(可以在25-50微米的范围内)。
      自由漂浮法:收集24孔中的切片。 在4℃下储存在PBS(pH 7.4,0.1M)中。 如果可能,立即工作。 否则,在-20°C下储存在冷冻保护剂(溶液6)中

  2. 免疫组织化学(免疫组化,自由浮动法)
    注意:如果切片在切割后立即安装到载玻片上,则每个载玻片需要约0.3ml工作溶液。这种方法将帮助你保存一些抗体。缺点是抗体可能不如自由浮动法中有效地穿透组织。此外,幻灯片很容易变干。
    对于自由浮动法,每孔需要〜0.5ml。对于30μM厚的切片,约7-8个切片可以在孔内进行相同的处理。使用具有非常细尖的滴管排出溶液。确保在洗涤/溶液更换期间不会损坏部件。
    1. 开始用PBS洗涤(3次,每次5分钟)
    2. 用含有0.25%Triton-100的正常驴血清封闭组织。室温(RT)下1小时
    3. 在封闭溶液中制备一抗。
      *短暂涡旋稀释抗体。
      第一抗体孵育在4℃下进行过夜。用石蜡膜包裹孔将防止脱水。为了立即的结果,可以在室温下孵育〜2-3小时。建议进行夜间孵育以获得足够的抗体孵育
    4. 从冰箱取出,让它在室温下〜1小时。
    5. 用PBS清洗(3次,每次5分钟)
    6. 二抗在RT下孵育1-2小时
    7. 孵育后,用PB(NOT PBS!)洗涤3次,每次5分钟
    8. 将部件小心地安装到载玻片上。 空气干燥,顶部覆盖(用锡箔或任何类型的防止光的盖子)。
    9. 在盖玻片前涂一滴Vectashield(Vector实验室)。
    10. 存放时,保持在-80°C

食谱

  1. 溶液1:磷酸盐缓冲液(PB)(0.2M储液,pH 7.4)
    一碱性NaP
    ddH 2 O(双蒸)250ml
    NaH 2 2 PO 4 6.9g
    二碱价
    ddH <2> O 1 L
    NaHPO 4 28.4g
    在〜100 ml的单碱性NaP中,开始添加二碱性NaP。 在加入二元碱的同时测量pH(可能需要约600-800ml)
    使用ddH 2 O将该0.2M储备溶液稀释1/2

  2. 解决方案2:4%多聚甲醛(PFA)用于经心脏灌注(每次使用新鲜,最多可存储3-4天)
    要制造1升PFA:
    加热900ml ddH 2 O至60℃(不超过70℃) 在通风橱中加入40g PFA粉末。 搅拌〜5分钟
    加入10滴2N NaOH直到溶液变澄清(可能需要几分钟)。 继续搅拌
    从热中取出并加入100ml的0.1M PB(Solution1)
    过滤并在4℃下储存过夜。 过滤是至关重要的; 否则,PFA碎片将堵塞血管
    将pH调节至4°C(测量前在此温度下需要标准化)
  3. 溶液3:0.9%盐水(〜500ml所需/大鼠脑)
    NaCl 9g/L的ddH 2 O·
    (可选)加入肝素以更好地循环。 推荐200单位/L的工作溶液
  4. 溶液4:用作冷冻保护剂的30%蔗糖的4%PFA 存储冷藏。
    向新鲜制备的4%PFA(溶液2)中加入30%(g)蔗糖
  5. 溶液5:磷酸盐缓冲盐水(PBS)
    0.01M磷酸钠在0.9%盐水中的溶液 做5 L
    将45g NaCl,250ml的0.2M PB(溶液1)加入4.5L的ddH 2 O中。
    (可选,但可能非常有助于灌注)加入肝素(200单位/L工作溶液),以防止血液凝固。
  6. 解决方案6.冷冻保护剂(用于长期保存切片)
    50%0.05M PB(稀释0.2M PB原液)
    30%乙二醇(无RNA酶)
    20%甘油(无RNA酶)
  7. 含有0.25%Triton-100的正常驴血清 对于1ml等分试样,
    25μl10%Triton
    875μlPBS
    100μl正常驴血清
    *脑组织不要超过1%triton。
  8. 一级抗体
    c-Fos
    电弧
    1μl在1,000μl封闭溶液中
  9. 次级抗体
    c-Fos IHC,Alexa antimouse Fluor 488,1:500稀释。
    Arc IHC,Cy3-缀合的驴抗兔二抗,1:500稀释 在PBS中稀释抗体(非封闭溶液)

致谢

该协议改编自Kim和Thompson(2011)。

参考文献

  1. Kim,S。和Thompson,R. F.(2011)。 c-Fos,Arc和stargazin在大鼠眼线调节中的表达。 Behav Neurosci 125(1):117-123。
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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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