All tags IHC protocols BrdU 免疫組織染色プロトコール

BrdU 免疫組織染色プロトコール

BrdU は、増殖細胞の検出ツールとして広く用いられています。ただし抗体で検出するのはタンパク質ではなくヌクレオチドなので、工夫が必要です。

PDF 版はこちら（英語）

チミジン・アナログであるブロモデオキシウリジン（BrdU）は、細胞周期の S 期において新たに合成された DNA に取り込まれます。こうして BrdU が取り込まれた（BrdU でラベルされた）DNA は、抗 BrdU 抗体で検出することができるため、抗 BrdU 抗体は DNA 複製を行う増殖細胞の検出ツールとなり、増殖細胞数の計測や形態の観察などの経時的実験系や、増殖細胞特異的な抗原の解析など、多様な実験に応用できます。また in vitro と in vivo、どちらのにも使用できます。

増殖細胞の識別には BrdU に加え、Ki67 や Doublecortin などとの多重染色が有効です。また細胞の分化マーカーとの多重染色を行なうことにより、より詳細な解析を行なうことができます。例えば NeuN との多重染色による分化直後の成熟ニューロンの識別などです。多重染色には蛍光標識済み抗体や Alexa Fluor® 標識済み二次抗体が便利です。

BrdU の検出には、免疫組織染色の他、イムノアッセイ、免疫蛍光細胞染色、フローサイトメトリーなど、さまざまなフォーマットで行なわれます。アブカムは各アプリケーションに対応した各種 BrdU 検出キット製品を各種取り揃えています。

以下に in vitro および in vivo の BrdU ラベリンク、抗体が BrdU と反応しやすくするための DNA 変性処理（DNA hydrolysis）、免疫組織染色による BrdU の検出について、プロトコールをまとめました（英文）。ぜひご活用ください。


BrdU labeling: in vitro labeling of cells with BrdU

  1. Prepare a 10 mM stock solution of BrdU (ab142567) by dissolving 3 mg of BrdU in 1 mL water.
  2. Dilute the 10 mM BrdU stock solution in cell culture medium to make a 10 µM BrdU labeling solution.
  3. Filter the 10 µM BrdU labeling solution through a 0.2 µm filter under sterile conditions.
  4. Remove the existing culture medium from the cells and replace with 10 µM labeling solution.
  5. Incubate the cells in the BrdU labeling solution for 1–24 hours at 37ºC in a CO2 incubator.

    Note: BrdU incubation time depends on how rapidly the cells divide. Primary cells may need up to 24 hours, while rapidly proliferating cell lines may only need one hour. The exact time required to achieve the optimal signal-to-noise ratio should be optimized.
  6. Remove the BrdU labeling solution from the cells and wash twice in PBS for about 5 seconds per wash.
  7. Wash three more times with PBS for two minutes each.
  8. Fix and permeabilize cells according to standard immunocytochemistry (ICC) protocols. However, before proceeding with immunostaining, refer to the DNA hydrolysis step below.


​BrdU labeling: in vivo labeling with BrdU

There are several methods for labeling cells in vivo with BrdU. Two commonly used methods are intraperitoneal injection and oral administration.

Intraperitoneal injection

  1. Dilute BrdU in PBS to make a sterile solution of 10 mg/mL.
  2. For mice, as a general rule, inject the BrdU solution to a concentration of 100 mg/kg.
  3. After treatment with BrdU, the animals can be sacrificed according to your lab's approved procedures.
  4. Fix and process tissue according to standard immunohistochemistry (IHC) protocols. However, before continuing with immunostaining, refer to the DNA hydrolysis step below.

Note: BrdU incorporation into rapidly dividing tissues, such as the small intestine, will be detectable as soon as 30 minutes post-injection. However, for most tissue you may need to wait up to 24 hours. The exact treatment time and dosage will need to be optimized for your tissue of interest.

Oral administration

Oral administration of BrdU is a non-invasive procedure and therefore useful for extended BrdU administration, although it may introduce variability into experiments due to lack of control over an animal’s water consumption.

  1. Dilute BrdU to 0.8 mg/mL in drinking water. Prepare this fresh and change daily.
  2. After treatment with BrdU, the animals can then be sacrificed according to standard protocols.
  3. Fix and process tissue according to standard IHC protocols. However, before immunostaining refer to the DNA hydrolysis step below.

Note: for mice, 225 mg/kg per day of BrdU (calculated by measuring water consumption volumes per animal) should achieve sufficient BrdU labeling. However, the exact dose should be optimized for individual experimental conditions.


​DNA hydrolysis

Preparation
Sodium borate buffer: 3.8g sodium borate (MW=381.4) + 100 ml distilled water. Adjust pH with NaOH.

Cells

  1. Incubate cells in 1–2.5 M HCL for 10 minutes to 1 hour at room temperature. The exact HCl concentration and incubation time should be optimized for your experiment. If using a shorter incubation time, incubating at 37oC may be more effective than room temperature.
  2. Optional step: remove the HCl and neutralize with 0.1 M sodium borate buffer pH 8.5 for 30 minutes at room temperature.
  3. Wash three times in PBS.
  4. Continue with immunostaining according to standard immunocytochemistry (ICC) protocols.


Tissue sections

Note: if using paraffin-embedded sections, ensure they are de-waxed before proceeding. Read our deparaffinization protocol here.

  1. Incubate sections in 1–2 M HCL for 30 minutes to 1 hour. The exact HCl concentration and incubation time should be optimized for your experiment. If using a shorter incubation time, incubating at 37oC may be more effective than room temperature.
  2. Optional step: neutralize sections by incubating in 0.1 M sodium borate buffer pH 8.5 for 10 minutes at room temperature.
  3. Wash three times in PBS for about 5 seconds per wash.
  4. Continue with immunostaining according to standard IHC protocols.

Note: some researchers have reported that they don’t perform the HCl hydrolysis step and simply perform heat-induced epitope retrieval before continuing with immunostaining. 


Co-staining with anti-BrdU

BrdU can be used in conjunction with other antibodies to identify proliferating and newly differentiated neurons.  See below for our suggestions.

Ki67
A cellular marker for proliferation, the Ki67 protein is present in cells at cycle phases G1, S, G2 and M, but absent in resting (G0) cells. Ki67 antibodies can be used instead of, or in conjunction with, BrdU to label proliferating neurons.

Read more about our knockout-validated Ki67 antibodies.

Doublecortin
A microtubule-associated phosphoprotein expressed by immature neurons. Doublecortin antibodies can be used in conjunction with BrdU to identify immature post-mitotic neurons.

Browse our doublecortin antibodies.

NeuN
​A marker of mature neurons that can be used in conjunction with BrdU staining to identify newly differentiated neurons.

Read about the advantages of our NeuN RabMAb® antibody 


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