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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。
There are several methods for labeling cells in vivo with BrdU. Two commonly used methods are intraperitoneal injection and oral administration.
Intraperitoneal injection
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.
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.
Preparation
Sodium borate buffer: 3.8g sodium borate (MW=381.4) + 100 ml distilled water. Adjust pH with NaOH.
Cells
Tissue sections
Note: if using paraffin-embedded sections, ensure they are de-waxed before proceeding. Read our deparaffinization protocol here.
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.
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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