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Cell Cycle Analysis in the Vertebrate Brain Using Immunolabeled Fresh Cell Nuclei
在脊椎动物脑部采用荧光标记新鲜细胞核进行细胞周期分析   

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Abstract

Flow cytometry, a standard technique used for quantitative analysis of isolated cells, is routinely employed by immunologists and oncologists to study DNA content, protein expression, and other functional parameters in blood and tumor cells. Unfortunately, the use of this technique by neurobiologists has been hampered by the complexity of the nervous system, whose constituting cells can hardly be dissociated to obtain samples of sufficient quality. We have developed a simplified and quick method to purify and immunolabel cell nuclei with high sensitivity and low background. Our protocol allows the discrimination of single nuclei from doublets and larger aggregates, obtaining low coefficients of variation for cell cycle analysis with propidium iodide. In addition, due to the reduced sample handling this method has high recovery and good reproducibility. As an example, in this protocol we describe the isolation of cell nuclei from adult cerebral cortex, which are subsequently immunostained with antibodies against NeuN (a general neuronal marker) and EGR1 (an early response gene expressed by functionally active neurons), and subjected to flow cytometric gating and analysis. Nevertheless, the protocol can also be applied to other neural tissues from adult and embryonic brain.

Keywords: Flow cytometry(流式细胞仪), DNA content(DNA含量), Ploidy(倍性), Tetraploid neuron(四倍体细胞), Diploid neuron(二倍体细胞)

Materials and Reagents

  1. Frozen tissue samples (adult mouse hemicortex)
  2. Phosphate-buffered saline (PBS)
  3. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  4. Protease inhibitors cocktail tablets (cOmplete, Mini, EDTA-free) (Roche Diagnostics, catalog number: 11 836 170 001 )
  5. Bovine serum albumin (BSA) (stock 30 mg/ml in PBS) (Sigma-Aldrich, catalog number: A4503 )
  6. Calf serum (Life Technologies, catalog number: 16170-086 )
  7. Primary antibodies:
    Mouse Anti-NeuN antibody (clone A60) (EMD Millipore, catalog number: mab377 )
    Rabbit anti-EGR1 antibody (clone T.126.1) (Thermo Fisher Scientific, catalog number: MA5-15009 )
  8. Secondary antibodies:
    Alexa Fluor 647 Goat Anti-Mouse IgG (H + L) Antibody (Life Technologies, catalog number: A21236 )
    Alexa Fluor 488 Donkey Anti-Rabbit IgG (H + L) Antibody (Life Technologies, catalog number: A21206 )
  9. Propidium iodide (stock 1 mg/ml, prepared in autoclaved water) (Sigma-Aldrich, catalog number: P4170 )
  10. RNase A (stock 1 mg/ml, inactivated by boiling as indicated by the manufacturer) (Sigma-Aldrich, catalog number: R5000 )
  11. DAPI (stock 100 μg/ml, prepared in autoclaved water) (Sigma-Aldrich, catalog number: D9542 )

Equipment

  1. 15 ml tube
  2. 1.5 ml Eppendorf tube
  3. 7 ml Dounce homogenizer (WHEATON, model: 357542 )
  4. Microscope
  5. Refrigerated centrifuge
  6. Autoclaved 40 μm nylon filters
  7. Emission filters: BP 530/30, BP 616/23, BP 660/20, APC, FITC
  8. FACSAria cytometer (BD Biosciences) equipped with a double argon (488 nm) and helium-neon (633 nm) laser

Software

  1. FACSDiva (BD Biosciences)
  2. Modfit LT software (Verity Software)

Procedure

  1. Cell nuclei isolation
    1. Prepare homogenization buffer containing PBS, 0.1% Triton X-100, and one tablet of protease inhibitors cocktail per 10 ml of buffer.
      Note: Keep buffers and sample on ice during processing.
    2. Transfer one mouse hemicortex (or an equivalent mass of adult nervous tissue) to a Dounce homogenizer containing 3 ml of homogenization buffer. The tissue is then homogenized on ice with the “loose” and “tight” pestles, as indicated by the manufacturer.
      Note: Volumes can be scaled according to sample size and complexity (for instance, 1 ml homogenization buffer for one telencephalic vesicle from an E17 embryo or one adult murine hippocampus). Several tissues can be processed in parallel, this will allow to get aliquots for control immunostainings (see step B2).
    3. Collect the sample and add 1.5 ml of PBS-0.1%Triton X-100 (final volume 4.5 ml).
    4. Centrifuge at 200 x g for 1.5 min at 4 °C to remove undissociated tissue (mostly blood vessels).
      Optional: Here and in subsequent centrifugation steps the sample can be aliquoted in 1.5 ml Eppendorf tubes to improve the visualization of the pellets.
    5. Discard pellet and transfer the supernatant to a 15 ml tube.
      Note: At this step you can remove a small aliquot of the sample, which should be labeled for at least 1 min with either DAPI (100 ng/ml) or propidium iodide (25 μg/ml) to check it under the microscope (Figure 1, Homog.).
    6. Add ice-cold PBS to a final volume of 12 ml.
      Note: Volumes can be scaled according to sample size and tissue structure. For instance, for embryonic tissues the volume can be reduced due to their low complexity (one telencephalic vesicle from an E17 embryo can be washed in 1.5 ml PBS). For one adult hippocampus 5 ml PBS are required.
    7. Centrifuge at 400 x g for 4 min at 4 °C.
    8. Discard supernatant and carefully add PBS to the pellet (total volume of 1-1.2 ml).
      Notes:
      1. CRITICAL STEP: Do not disturb the pellet nor try to resuspend it at this point. It is crucial not to disturb the cell nuclei to maintain their integrity!
      2. At this step you can remove a small aliquot of the supernatant and, after adding either DAPI (100 ng/ml) or propidium iodide (25 μg/ml), check under the microscope that cell nuclei are absent (Figure 1, Supernat.).
    9. Maintain the cell nuclei pellet on ice for 20-30 min.
    10. Resuspend the pellet by gently swirling of the vial (until no visible lumps can be observed), and then by pipetting up and down.
      Notes:
      1. At this step remove a small aliquot of the sample and, after adding either DAPI (100 ng/ml) or propidium iodide (25 μg/ml) check the quality of cell nuclei under the microscope (Figure 1, Sample).
      2. Samples can be stored at 4 °C for a few hours until immunostaining and for 24 h for cell cycle analysis.
    11. If immunostaining is not required proceed to step B6.


      Figure 1. Microscopic images of the aliquots obtained during the cell nuclei isolation procedure. Aliquots of the samples obtained in steps A5 (Homog.), A8 (Supernat.), and A10 (Sample) stained with DAPI (blue).

  2. Fresh/unfixed nuclei immunostaining
    1. To block nonspecific immunostaining, add 4-5 μl calf serum and 6.4-8 μl BSA (30 mg/ml) to 400-500 μl of the cell nuclei suspension obtained in step A10. Mix by inversion and proceed without further delay.
    2. Prepare three control samples (400-500 μl each) by pooling small aliquots of the cell nuclei suspensions obtained in step A10, and block nonspecific immunostaining as indicated above:
      1. Secondary antibody control (CONT).
      2. Positive control for NeuN (POSITIVE NEUN+).
      3. Positive control for EGR1 (POSITIVE EGR1+).
        Note: This allows to keep enough volume from the cell nuclei suspensions obtained in step A10 to perform at least two different immunostainings (even for small tissues such as the hippocampus).
    3. Add primary antibody/antibodies against nuclear proteins and mix by inversion:
      1. Secondary antibody control: No primary antibody.
      2. Positive control for NeuN: 0.6 μl of mouse anti-NeuN in 500 μl of sample.
      3. Positive control for EGR1: 1 μl of rabbit anti-EGR1 in 500 μl of sample.
      4. Sample: 0.6 μl of mouse anti-NeuN and 1 μl of rabbit anti-EGR1 in 500 μl of sample.
        Note: Dilutions for other primary antibodies should be empirically optimized (usually the same or double concentration as used for immunohistochemistry should be a good choice). If possible, select antibodies known to recognize unfixed/native epitopes (i.e. validated for immunoprecipitation).
    4. Without washing, add 1/500 dilution of secondary antibody/antibodies to all the samples (including the controls) and mix by inversion.
      Note: We combine primary and secondary antibodies to avoid repeated washing steps that could result in loss of material. Be sure that the fluorophores you use are both excitable and detectable by the cytometer and that they are compatible with the excitation/emission spectra of each other, as well as with that of propidium iodide.
    5. Incubate over night at 4 °C in the dark (without shaking).
      Note: Alternatively, samples can be incubated for 2 h at room temperature.
    6. Carefully resuspend the sedimented nuclei and, without washing, filter the sample through a 40 μm autoclaved (i.e. DNase-free) nylon filter.
      Note: At this step remove a small aliquot of the sample and, after adding either DAPI (100 ng/ml) or propidium iodide (25 μg/ml), check under the microscope the immunstaining signals (Figure 2).


      Figure 2. Microscopic images of the aliquots of cell nuclei obtained after immunostaining

    7. Add propidium iodide to the samples to a final concentration of 25-50 μg/ml and DNase-free RNase A to a final concentration of 25 μg/ml.

  3. Flow cytometric gating and analysis
    1. Analyze the samples using a flow cytometer.
      Note: In our case, we use a FACSAria cytometer equipped with a double argon (488 nm) and helium-neon (633 nm) laser. Emission filters: BP 530/30 (for Alexa 488), BP 616/23 (for propidium iodide), and BP 660/20 (for Alexa 647). Data are collected using a linear digital signal process. For data analysis, we use FACSDiva and Modfit LT software.
    2. Localize the nuclei population in the (forward scatter) FSC vs. (side scatter) SSC plot, and create a first gate: P1 (Figure 3A).
    3. Simultaneously eliminate the debris (i.e. the population unstained for IP) by creating a P2 gate in the Propidium Iodide-A vs. Propidium Iodide-H plot (Figure 3B). This should greatly improve the identification of the nuclei in the FCS-A vs. SSC-A plot (Figure 3C), as well as the fine-tuning of P1 (Figure 3A).
    4. Select the singlet population and exclude doublets and clumps by gating (P3) on the Propidium Iodide-A vs. Propidium Iodide-H plot (Figure 3D) (see Figure 4 for further explanation).


      Figure 3. Contour plots and gated scheme used to detect nuclei from cerebral cortex of adult mice


      Figure 4. Scheme explaining the procedure used for doublet discrimination. As propidium iodide-labeled nuclei transit through the laser beam, the fluorescence signal is converted into a voltage pulse defined by its height (H), width (W), and area (A). The integrated area of the signal (Propidium iodide-A) is proportional to the DNA content. The Propidium Iodide-W variable directly depends on the time spent by the particle crossing the laser beam and the Propidium Iodide-H reflects the maximal intensity of fluorescence signal. A 4C singlet (tetraploid nucleus) can be discriminated from a doublet (two diploid nuclei adhered to each other) due to the higher H/A ratio and lower W/A ratio of its fluorescence signal (adapted from López-Sánchez and Frade, 2013).

    5. Confirm the correct exclusion of doublets in the selected population by checking the Propidium Iodide-A vs. Propidium Iodide-W plot and adjust P3 if needed. The population with 4C DNA content and high Propidium iodide-W should disappear (compare Figure 3E with Figure 3F). The resulted population is constituted by the singlets to be analyzed henceforth.
    6. In the control sample (CONT), determine the background threshold of fluorescence signals using emission filter APC (for NeuN) and emission filter FITC (for EGR1). The threshold can be determined representing individual signals in a histogram (Figure 5A-B) or simultaneously representing both signals on the same dot plot (Figure 6A).


      Figure 5. Histograms representing the fluorescence signals for NeuN and EGR1 in secondary antibody controls (CONT) for NeuN (A) and EGR1 (B), as well as sample populations (SAMPLE) immunostained for NeuN (C) and EGR1 (D)


      Figure 6. Dot plots representing the fluorescence signals for NeuN and EGR1 in secondary antibody control (A), positive control for NeuN (B), positive control for EGR1 (C), and sample populations (D)

    7. Select positive populations in the individual positive controls (Figure 5C-D and Figure 6B-C). This allows to identify the double-labeled population (Figure 6D).
      Note: Sometimes nuclei of interest have specific characteristics of size or complexity that can be used for defining specific nuclei populations. For instance, most of the NeuN positive nuclei from adult mice has higher SSC-A than the negative ones (Figure 7). This allows to improve the correct gating of this population.


      Figure 7. Dot plots of NeuN vs. SSC-A signals

    8. Finally, analyze the DNA content of the population/s of interest in each sample.
      Note: This method is useful for both cell cycle analysis in proliferating tissues (Figure 8A-B) and quantification of DNA content in NeuN+ nuclei (Figure 8C-D).


      Figure 8. DNA content analysis. Typical histogram of DNA content from a proliferating tissue (A), and analysis of its cell cycle phases quantified with Modfit software (B). Representative histograms showing non-neuronal (C) and neuronal (D) cell nuclei from adult mouse brain. 2C: diploid, 4C: tetraploid.

Notes

  1. Similar results have been obtained with nervous tissue from other vertebrates, including embryonic and post hatch chicken (Lopez-Sanchez and Frade, 2013) and human (unpublished data).

Acknowledgments

This protocol is adapted from a previous paper by Lopez-Sanchez and Frade (2013). The experimental work was supported by grants from the “Ministerio de Ciencia e Innovación” (BFU2009-07671 and SAF2012-38316) and “Fundación Areces” (CIVP16A1815). Noelia López-Sánchez acknowledges a JAE-Doc contract (JAEDoc026, 2008 call) from the CSIC program “Junta para la Ampliación de Estudios”, co-funded by European Social Fund.

References

  1. López-Sánchez, N. and Frade, J. M. (2013). Genetic evidence for p75NTR-dependent tetraploidy in cortical projection neurons from adult mice. J Neurosci 33(17): 7488-7500.

简介

Flow cytometry, a standard technique used for quantitative analysis of isolated cells, is routinely employed by immunologists and oncologists to study DNA content, protein expression, and other functional parameters in blood and tumor cells. Unfortunately, the use of this technique by neurobiologists has been hampered by the complexity of the nervous system, whose constituting cells can hardly be dissociated to obtain samples of sufficient quality. We have developed a simplified and quick method to purify and immunolabel cell nuclei with high sensitivity and low background. Our protocol allows the discrimination of single nuclei from doublets and larger aggregates, obtaining low coefficients of variation for cell cycle analysis with propidium iodide. In addition, due to the reduced sample handling this method has high recovery and good reproducibility. As an example, in this protocol we describe the isolation of cell nuclei from adult cerebral cortex, which are subsequently immunostained with antibodies against NeuN (a general neuronal marker) and EGR1 (an early response gene expressed by functionally active neurons), and subjected to flow cytometric gating and analysis. Nevertheless, the protocol can also be applied to other neural tissues from adult and embryonic brain.

关键字:流式细胞仪, DNA含量, 倍性, 四倍体细胞, 二倍体细胞

材料和试剂

  1. 冷冻组织样品(成年小鼠外胚层)
  2. 磷酸盐缓冲盐水(PBS)
  3. Triton X-100(Sigma-Aldrich,目录号:T8787)
  4. 蛋白酶抑制剂混合物片剂(cOmplete,Mini,无EDTA)(Roche Diagnostics,目录号:11 836 170 001)
  5. 牛血清白蛋白(BSA)(PBS中的储备液为30mg/ml)(Sigma-Aldrich,目录号:A4503)
  6. 小牛血清(Life Technologies,目录号:16170-086)
  7. 主要抗体:
    小鼠抗NeuN抗体(克隆A60)(EMD Millipore,目录号:mab377)
    兔抗EGR1抗体(克隆T.126.1)(Thermo Fisher Scientific,目录号:MA5-15009)
  8. 次级抗体:
    Alexa Fluor 647山羊抗小鼠IgG(H + L)抗体(Life Technologies,目录号:A21236)
    Alexa Fluor 488驴抗兔IgG(H + L)抗体(Life Technologies,目录号:A21206)
  9. 碘化丙啶(储备液1mg/ml,在高压灭菌水中制备)(Sigma-Aldrich,目录号:P4170)
  10. RNA酶A(储备液1mg/ml,如制造商所示通过煮沸灭活)(Sigma-Aldrich,目录号:R5000)
  11. DAPI(储存在100μg/ml,在高压灭菌水中制备)(Sigma-Aldrich,目录号:D9542)

设备

  1. 15 ml管
  2. 1.5 ml Eppendorf管
  3. 7ml Dounce匀浆器(WHEATON,型号:357542)
  4. 显微镜
  5. 冷冻离心机
  6. 高压灭菌的40μm尼龙过滤器
  7. 发射滤光片:BP 530/30,BP 616/23,BP 660/20,APC,FITC
  8. 装备有双氩(488nm)和氦 - 氖(633nm)激光的FACSAria细胞计数器(BD Biosciences)

软件

  1. FACSDiva(BD Biosciences)
  2. Modfit LT软件(Verity软件)

程序

  1. 细胞核分离
    1. 制备包含PBS,0.1%Triton X-100和一片蛋白酶抑制剂混合物/10ml缓冲液的匀浆缓冲液。
      注意:在处理过程中将缓冲液和样品保存在冰上。
    2. 转移一个小鼠的hemicortex(或等同质量的成年神经组织)到一个含有3毫升匀浆缓冲液的Dounce匀浆器。 然后将组织与"松散"在冰上匀浆 "紧"杵,如制造商所示。
      注意:体积可以根据样品大小和复杂性(例如,对于来自E17胚胎或一个成年鼠海马的一个端脑泡的1ml匀浆缓冲液)缩放。可以平行处理几种组织,这将允许获得等分试样用于对照免疫染色(参见步骤B2)。
    3. 收集样品,加入1.5ml PBS-0.1%Triton X-100(终体积4.5ml)
    4. 在4℃下以200×g离心1.5分钟以除去未解离的组织(主要是血管)。
      可选:在这里和随后的离心步骤中,样品可以等分在1.5ml Eppendorf管中以改善沉淀的可视性。
    5. 弃去沉淀并将上清液转移到15ml管中 注意:在这一步,你可以删除一小部分的样品,应该用DAPI(100 ng/ml)或碘化丙啶(25μg/ml)标记至少1分钟,检查它在显微镜(图1,Homog。)。
    6. 加入冰冷PBS至终体积12 ml 注意:体积可以根据样品大小和组织结构进行缩放。例如,对于胚胎组织,体积可以由于其低复杂性而降低(来自E17胚胎的一个端脑泡可以在1.5ml PBS中洗涤)。对于一个成年海马,需要5ml PBS。
    7. 在4℃下以400xg离心4分钟。
    8. 弃去上清液,小心地向沉淀中加入PBS(总体积为1-1.2ml) 注意:
      1. 关键步骤:不要打扰沉淀,或尝试在此时重新悬浮沉淀。 至关重要的是不要打扰细胞核以维持其完整性!
      2. 在该步骤中,可以除去上清液的一小部分,并且在加入DAPI(100ng/ml)或碘化丙啶(25μg/ml)后,在显微镜下检查细胞核不存在(图1, Supernat。)。
    9. 保持细胞核丸在冰上20-30分钟。
    10. 通过轻轻旋转小瓶(直到没有可观察到的块状物),然后通过上下吹吸来重悬沉淀。
      注意:
      1. 在该步骤中,除去样品的一小部分,并且在加入DAPI(100ng/ml)或碘化丙啶(25μg/ml)后,在显微镜下检查细胞核的质量(图1,样品)。
      2. 样品可以在4℃下储存几小时直至免疫染色,并且在细胞周期分析中储存24小时。
    11. 如果不需要免疫染色,则进行步骤B6

      图1.在细胞核分离过程中获得的等分试样的显微图像。 在步骤A5(Homog。),A8(Supernat。)和A10(Sample)中获得的样品的等分试样用DAPI(蓝色)染色。

  2. 新鲜/未固定细胞核免疫染色
    1. 为了阻断非特异性免疫染色,向400-500μl在步骤A10中获得的细胞核悬液中加入4-5μl小牛血清和6.4-8μlBSA(30mg/ml)。 通过倒置混合,并且进一步延迟。
    2. 通过汇集步骤A10中获得的细胞核悬液的小等分试样,并且阻断如上所示的非特异性免疫染色,制备三个对照样品(每个400-500μl):
      1. 二抗抑制(CONT)。
      2. NeuN的阳性对照(POSITIVE NEUN +)。
      3. 正向控制EGR1(正向EGR1 +)。
        注意:这允许保持足够的体积与在步骤A10中获得的细胞核悬液进行至少两种不同的免疫染色(即使对于小组织如海马)。
    3. 添加抗核蛋白的一抗/抗体,通过倒置混合:
      1. 二抗抑制:无一抗。
      2. NeuN的阳性对照:在500μl样品中的0.6μl小鼠抗NeuN
      3. EGR1的阳性对照:在500μl样品中1μl兔抗-EGR1
      4. 样品:在500μl样品中的0.6μl小鼠抗NeuN和1μl兔抗-EGR1 注意:其他初级抗体的稀释度应该根据经验进行优化(通常与免疫组织化学使用的相同或两倍的浓度应该是一个不错的选择)。如果可能,选择已知识别未固定/天然表位的抗体(即经过免疫沉淀验证)。
    4. 不洗涤,向所有样品(包括对照)中加入1/500稀释的二抗/抗体,并通过倒置混合 注意:我们将主抗体和次抗体组合以避免可能导致材料损失的重复清洗步骤。确保您使用的荧光团都是可激发的,可以通过细胞仪检测,并且它们与彼此的激发/发射光谱以及与碘化丙啶的激发/发射光谱兼容。
    5. 在4℃下在黑暗中孵育过夜(无振荡) 注意:或者,样品可以在室温下孵育2小时。
    6. 小心地重悬沉淀的核,并且在不洗涤的情况下,通过40μm高压灭菌(即不含DNase的)尼龙过滤器过滤样品。
      注意:在该步骤中,除去样品的一小部分,并且在加入DAPI(100ng/ml)或碘化丙啶(25μg/ml)后,在显微镜下检查免疫染色信号(图2)/em>


      图2.免疫染色后获得的细胞核的等分试样的显微图像

    7. 向样品中加入碘化丙啶至终浓度为25-50μg/ml和无DNA酶的核糖核酸酶A至终浓度为25μg/ml。

  3. 流式细胞仪门控和分析
    1. 使用流式细胞仪分析样品。
      注意:在我们的情况下,我们使用配备双氩(488nm)和氦氖(633nm)激光的FACSAria细胞仪。 发射过滤器:BP 530/30(对于Alexa 488),BP 616/23(对于碘化丙啶)和BP 660/20(对于Alexa 647)。 使用线性数字信号处理收集数据。 对于数据分析,我们使用FACSDiva和Modfit LT软件。
    2. 在(前向散射)FSC对(侧向散射)SSC图中定位核子总体,并创建第一个门:P1(图3A)。
    3. 通过在碘化丙锭-A对碘化丙锭 - 丙酸图中产生P2闸门(图3B),同时消除碎片(即IP未污染的群体)。这将大大改善在FCS-A对SSC-A图(图3C)以及P1的微调(图3A)的核的鉴定。
    4. 通过在碘化丙锭-A对碘化丙锭-H图(图3D)上的门控(P3)选择单峰群体并排除双峰和团块(参见图4以进一步解释)。


      图3.用于检测成年小鼠大脑皮质核的轮廓图和门控方案


      图4.解释用于双重鉴别的程序的方案。由于碘化丙啶标记的核通过激光束,荧光信号被转换为由其高度(H),宽度W)和面积(A)。信号的积分面积(碘化丙啶-A)与DNA含量成比例。碘化丙锭的变量直接取决于粒子穿过激光束所花费的时间,碘化丙锭反映荧光信号的最大强度。由于其荧光信号的更高的H/A比和更低的W/A比,可以将4C单倍体(四倍体核)与双峰(两个二倍体核彼此粘附)区分开来(改编自López-Sánchez和Frade,2013 )。

    5. 通过检查碘化丙锭-A对碘化丙锭 - 碘图,确认所选群体中双联体的正确排除,如果需要,调整P3。具有4C DNA含量和高碘化丙啶-W的群体应消失(将图3E与图3F比较)。得到的总体由以下要分析的单子构成。
    6. 在对照样品(CONT)中,使用发射滤光片APC(对于NeuN)和发射过滤器FITC(对于EGR1)确定荧光信号的背景阈值。可以确定表示直方图(图5A-B)中的单个信号或同时表示相同点图上的两个信号(图6A)的阈值。


      图5.代表NeuN(A)和EGR1(B)的二次抗体对照(CONT)中NeuN和EGR1的荧光信号的直方图,以及NeuN(C)和EGR1(B)免疫染色的样品群体(SAMPLE) D)


      图6.代表第二抗体对照(A),NeuN(B)阳性对照,EGR1(C)阳性对照和样品群体(D)的NeuN和EGR1的荧光信号的点图。

    7. 在个别阳性对照中选择阳性群体(图5C-D和图6B-C)。这允许鉴定双标记群体(图6D)。
      注意:有时,感兴趣的核具有可用于定义特定细胞核群体的大小或复杂性的特定特征。例如,来自成年小鼠的大多数NeuN阳性细胞核具有比阴性细胞更高的SSC-A(图7)。这可以改善此群体的正确选通。


      图7. NeuN与SSC-A信号的点图

    8. 最后,分析每个样品中感兴趣的群体的DNA含量。
      注意:该方法对于增殖组织中的细胞周期分析(图8A-B)和NeuN +细胞核中DNA含量的定量(图8C-D)都是有用的。


      图8.DNA含量分析。来自增殖组织的DNA含量的典型直方图(A),以及用Modfit软件(B)定量的其细胞周期相的分析。显示来自成年小鼠脑的非神经元(C)和神经元(D)细胞核的代表性直方图。 2C:二倍体,4C:四倍体

笔记

  1. 使用来自其他脊椎动物的神经组织,包括胚胎和孵化后的鸡(Lopez-Sanchez和Frade,2013)和人(未发表的数据)也获得了类似的结果。

致谢

该协议改编自Lopez-Sanchez和Frade(2013)的前一篇文章。 实验工作由"Ministerio de Ciencia eInnovación"(BFU2009-07671和SAF2012-38316)和"FundaciónAreces"(CIVP16A1815)的赠款支持。 NoeliaLópez-Sánchez承认来自欧洲社会基金共同资助的CSIC计划"Junta para laAmpliaciónde Estudios"的JAE-Doc合同(JAEDoc026,2008年电话)。

参考文献

  1. López-Sánchez,N.和Frade,J. M.(2013)。 来自成人的皮质投影神经元中p75 NTR - 依赖性四倍体的遗传证据 小鼠。 J Neurosci 33(17):7488-7500。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Lopez-Sanchez, N. and Frade, J. M. (2013). Cell Cycle Analysis in the Vertebrate Brain Using Immunolabeled Fresh Cell Nuclei. Bio-protocol 3(22): e973. DOI: 10.21769/BioProtoc.973.
  2. López-Sánchez, N. and Frade, J. M. (2013). Genetic evidence for p75NTR-dependent tetraploidy in cortical projection neurons from adult mice. J Neurosci 33(17): 7488-7500.
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