FM 1-43 (N-(3-Triethylammoniumpropyl)-4-(4-(dibutylamino)styryl pyridinium dibromide)

神經(jīng)末梢熒光探針

產(chǎn)品簡(jiǎn)介：

FM 1-43，英文全名：N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide，是一種親脂的苯乙烯染料，是一種優(yōu)秀的膜探針用來(lái)鑒定活躍的放電神經(jīng)元和用于調(diào)研活動(dòng)依賴性的囊泡循環(huán)。這一水溶性的染料對(duì)細(xì)胞無(wú)毒，且在水溶液中基本無(wú)熒光，一旦插入細(xì)胞膜外層后發(fā)射強(qiáng)熒光。在活躍釋放神經(jīng)遞質(zhì)的神經(jīng)元中，FM 1-43內(nèi)在化進(jìn)入循環(huán)的突出囊泡，神經(jīng)末梢被明亮染色。成像檢測(cè)前只需簡(jiǎn)單清洗去除非特異性得細(xì)胞表面膜染色。

產(chǎn)品特性：

1） CAS NO.：149838-22-2

2） 同義名：Pyridinium, 4-[2-[4-(dibutylamino)phenyl]ethenyl]-1-[3-(triethylammonio)propyl]-, dibromide

3） 分子式：C30H49Br2N3

4） 分子量：611.54

5） 純度：≥95%（HPLC）

6） 溶解性：溶于水

7） Ex/Em：510/626nm (MeOH); 480/598 (membrane-bound);

8）化學(xué)結(jié)構(gòu)圖：

保存條件: 

-20°C避光干燥保存，至少1年有效。

產(chǎn)品使用：

1. 儲(chǔ)存液配制

于實(shí)驗(yàn)前，將凍干粉置于室溫回溫至少20min，加入無(wú)菌水配制成10mM或其他濃度儲(chǔ)存液，比如，對(duì)于1mgFM 1-43（MW: 611.54）加入163μl DMSO,充分溶解后即得到10mM儲(chǔ)存液，根據(jù)單次用量分裝，≤-20℃凍存，避免反復(fù)凍融。

2. 染色方法（僅作參考）

應(yīng)用示例（來(lái)自文獻(xiàn)，僅作參考）

FM 1–43 Labeling and Unloading：

① FM 1–43儲(chǔ)存液(1000X):用水溶解FM1-43配制4mM儲(chǔ)存液。按20μL每管分裝保存在-20℃避光。如果希望標(biāo)記后固定樣本，選擇FM 1–43FX。

② HL-3 + 90 mM KCl溶液: 25 mM NaCl, 90 mM KCl, 10 mM NaHCO3, 5 mM HEPES, 30 mM sucrose, 5 mM threalose, 10 mM MgCl2, pH to 7.2.新鮮制備，超過(guò)2d后不可使用，4℃保存。加入CaCl2（1M標(biāo)準(zhǔn)液）以得到終濃度為1.5mM的CaCl2（或希望使用的其他濃度）。

③ 使用KCl刺激法用FM1-43標(biāo)記突觸小泡（Labeling of Synaptic Vesicles with FM 1–43 Using KCl Stimulation）：

◇FM 1–43標(biāo)記液: Add 1 μL FM 1–43 stock solution to 1 mL HL-3 plus 90 mM KCl solution with calcium (final concentration of FM 1–43 is 4 μM).

◇Incubate the dissected larva in FM 1–43 labeling solution by replacing the HL-3 solution without calcium. Do not add the solution on top of the larva but gently pipet the solution on the side of the larva. Start a timer; once you add the labeling solution, exocytosis and endocytosis are induced, and FM 1–43 labeling ensues. The incubation time will depend on your experiment, but a 1-min incubation leads to robust labeling in wild-type animals). Since some mutants are temperature sensitive, it may be necessary to perform experiments at high temperature (see Note 2 if this is the case).

◇Following stimulation, remove FM 1–43 labeling solution and wash five times over a total of 5–10 min with a generous amount of HL-3 solution without calcium to stop stimulating and to remove dye not internalized. Do not add the solution on top of the larva but gently pipet the solution on the side of the larva to avoid muscle damage. Gently perfuse the wash solution by pipeting up and down.

◇Image labeled vesicles on a confocal microscope with an ×40 water immersion lens and quantify intensity of labeling as described in Subheading 3.5.

Fig. FM 1–43 labeling of a third-instar larval fillet. (A)–(E) Third instar larval fillet incubated for 1 min in HL-3 with 4 μM FM 1–43, 90 mM KCl, and 1.5 mM calcium, washed, and the FM 1–43 imaged (D). The same preparation was then briefly fixed with 3.7% formaldehyde and labeled with mouse anti-DLG antibodies (28; 4F3 monoclonal antibody; Developmental Studies Hybridoma bank) used at 1:50 (B), (C), and (E) to reveal synaptic boutons at the NMJs and Alexa 635-conjugated phalloidin (Invitrogen cat. no. A34054) used at 0.001 unit/μL (A) and (C) to reveal muscles. (A)–(C) two hemisegments labeled with phalloidin (A) and DLG (B), merged in (C). The dashed line in (A) shows the ventral midline, and muscles commonly used in the genetic analysis of synaptic function are marked. (D) FM 1–43 labeling (before fixation of the preparation) of the NMJ in the boxed area on muscle 4 shown in (C). (E) The same synapse as shown in (D), labeled with DLG (see B) following fixation of the preparation.

注意事項(xiàng)：

1.      熒光染料都存在淬滅的問(wèn)題，保存和操作過(guò)程中注意避光。

2.      苯乙烯染料在水溶液中基本無(wú)熒光，最大發(fā)射波長(zhǎng)具pH依賴性。苯乙烯染料的光譜特征在甲醇或氯仿中測(cè)定。其在膜環(huán)境中的最大激發(fā)和最大發(fā)射波長(zhǎng)都會(huì)變短。最大激發(fā)波長(zhǎng)的差異通常在20nm，發(fā)射波長(zhǎng)的差異通常是80nm，但依具體的探針有所差異。

3.      FM^?是Molecular Probe公司的注冊(cè)商標(biāo)。

4.      為了您的安全和健康，請(qǐng)穿實(shí)驗(yàn)服并戴一次性手套操作。

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