上海信帆生物帶您了解：組織自發(fā)熒光淬滅劑 

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組織自發(fā)熒光淬滅劑 

描述：許多組織會(huì)產(chǎn)生可透過各種波長(zhǎng)濾光片的組織內(nèi)源性自發(fā)熒光，顯著干擾抗體標(biāo)記熒光觀察甚至導(dǎo)致熒光組化染色失敗。自發(fā)熒光淬滅試劑中的離子可用碰撞方式捕獲自發(fā)熒光光源分子發(fā)出的電子，阻止該電子從激發(fā)態(tài)回歸基態(tài)并阻止能量釋放，從而淬滅自發(fā)熒光。采用優(yōu)化的孵育時(shí)間，可以大限度地消除自發(fā)熒光而不明顯影響抗體標(biāo)記的熒光。

適用：各種組織、細(xì)胞免疫熒光染色的自發(fā)熒光消除。特別適用于神經(jīng)組織自發(fā)熒光淬滅。

儲(chǔ)存：4 ºC避光。

用法：

以下步驟在免疫熒光組化染色完畢之后(而非在熒光染色完畢之前)執(zhí)行。對(duì)特定的組織和細(xì)胞類型，必需優(yōu)化孵育時(shí)間以便大限度淬滅自發(fā)熒光而不明顯影響抗體標(biāo)記的熒光(步驟2)。仔細(xì)閱讀后面說明。

1. 吸去PBS或相應(yīng)洗滌緩沖液，用蒸餾水短暫沖洗組織切片或細(xì)胞培養(yǎng)板中的細(xì)胞。

2. 加入適量但充足的自發(fā)熒光淬滅劑覆蓋組織切片或瓶皿中的細(xì)胞。室溫10-90min。

3. 吸去自發(fā)熒光淬滅劑，用蒸餾水短暫沖洗。

4. 吸去蒸餾水，用PBS覆蓋組織切片或位于細(xì)胞培養(yǎng)板中的細(xì)胞。

5. 封片。建議使用抗熒光衰減封片劑。該封片劑可防止抗體標(biāo)記熒光衰退。

6. 熒光顯微鏡觀察。

說明：

1.不同物種不同類型的組織的自發(fā)熒光具有不同的特征，使用組織自發(fā)熒光淬滅劑的效果可能會(huì)有差別。另外，任何針對(duì)自發(fā)熒光的淬滅，將會(huì)在一定程度上降低抗體熒光強(qiáng)度。所幸的是該試劑對(duì)自發(fā)熒光的淬滅程度遠(yuǎn)遠(yuǎn)超出抗體熒光強(qiáng)度的降低，因而能在二者之間獲得較好的平衡。由于不很清楚的原因，本試劑消除腦脊髓神經(jīng)組織的自發(fā)熒光具有更好的效果。

2. 為獲得佳效果，必需優(yōu)化孵育時(shí)間以便大限度淬滅對(duì)某一特定組織的自發(fā)熒光而不明顯影響抗體標(biāo)記的熒光(步驟2)。重要的標(biāo)本應(yīng)在確定佳孵育時(shí)間之后使用本試劑。進(jìn)行優(yōu)化時(shí)，可取數(shù)張組織切片或位于培養(yǎng)皿中的細(xì)胞，在免疫熒光組化染色完畢之后加入組織自發(fā)熒光淬滅劑，孵育5、10、30、60、90分鐘等不同時(shí)間，沖洗后觀察熒光。如果組織自發(fā)熒光仍然很強(qiáng)，可延長(zhǎng)孵育時(shí)間；如果孵育時(shí)間小于10分鐘而熒光消退十分明顯，可將孵育時(shí)間減少為1-5分鐘，或者可取出少量的組織自發(fā)熒光淬滅劑加等份的雙蒸水稀釋，然后孵育10-90分鐘優(yōu)化。

3. 組織自發(fā)熒光淬滅劑必須在完成免疫熒光組化染色后使用，否則將嚴(yán)重降低抗體熒光。

AutoFluo Quencher

Description:

Many tissues can produce endogenous spontaneous fluorescence which can be observed through various wavelength filters, which can obviously interfere with antibody labeling fluorescence and even lead to the failure of fluorescence histochemical staining. The ions in the spontaneous fluorescence quenching reagent can capture the electrons emitted by the spontaneous fluorescent light source molecules in a collision mode, which prevents the electron from returning to the ground state and prevents the energy release from the excited state, thereby quenching the spontaneous fluorescence. By optimizing the incubation time, the fluorescence of the antibody could be eliminated and the fluorescence was not obviously affected by the spontaneous fluorescence. 

Application: spontaneous fluorescence elimination of various tissues and cells by immunofluorescence staining. In particular, the application of neural tissue spontaneous fluorescence quenching.

Storage at :  4 degrees C & keep away from light.

Usage:

The following steps are performed after the IHC staining (and not before the fluorescent staining). For specific tissues and cell types, it is necessary to optimize the incubation time in order to maximize the quenching of the fluorescence (step 2), which is not significantly affected by the antibody labeling. Read the instructions carefully.

1 suction to PBS or the corresponding washing buffer, using distilled water briefly washed tissue sections or cell culture plate in the cell.

2 adding adequate amount of spontaneous fluorescent quenching agent to cover the cells in tissue sections or bottles. Room temperature 10-90min.

3 to absorb the spontaneous fluorescence quenching agent, with distilled water briefly rinse.

4 suck the distilled water, cover the tissue sections with PBS or cells in the cell culture plate.

5 mounting. Anti sealing agents using the proposed fluorescence decay. The sealing agents can prevent the antibody labeled with fluorescent decay.

6 fluorescence microscope observation.

Note：

1. different types of tissues of different types of spontaneous fluorescence with different characteristics, the use of spontaneous fluorescence quenching agent effect may be different. In addition, any quenching of spontaneous fluorescence will reduce the fluorescence intensity of antibody to a certain extent. Fortunately, the quenching degree of the reagent is far more than the decrease of the fluorescence intensity of the antibody, so that a good balance between the two can be obtained. Because of the unclear reasons, this reagent eliminates the spontaneous fluorescence of the spinal cord nerve tissue and has a better effect.

2. in order to obtain the best results, it is necessary to optimize the incubation time in order to maximize the quenching of the spontaneous fluorescence of a particular tissue and not to affect the fluorescence (step 2). Important specimens should be used to determine the optimal incubation time. Optimize, the number of desirable tissue section or in the cells in a Petri dish, after immunohistochemistry after adding tissue fluorescence quenching agent, were incubated for 5, 10, 30, 60, 90 minutes of different time, observe the fluorescence after washing. If the tissue autofluorescence is still strong, can prolong the incubation time; if the incubation time is less than 10 minutes and fluorescence fade is very obvious, can reduce the incubation time for 1-5 minutes, or you can remove a small amount of tissue autofluorescence quenching agent with equal distilled water dilution, and then incubated for 10-90 minutes optimization.

3. tissue spontaneous fluorescent quenching agent must be used after the completion of immunohistochemical staining, otherwise it will seriously reduce the antibody fluorescence.

上海信帆生物帶您了解：組織自發(fā)熒光淬滅劑